Carotenoids for treating or preventing nausea

ABSTRACT

Methods and compositions comprising carotenoids for the treatment or prevention of nausea, e.g., chemotherapy-induced nausea and vomiting.

CLAIM OF PRIORITY

This application claims the benefit of U.S. Provisional Application Ser. No. 62/802,398, filed on Feb. 7, 2019. The entire contents of the foregoing are incorporated herein by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant No. AG043184 awarded by the National Institutes of Health. The Government has certain rights in the invention.

BACKGROUND

Chemotherapy-induced Nausea and Vomiting (CINV) affects 70-80% of patients undergoing chemotherapy and is a leading cause of discontinuation of cancer treatment.

SUMMARY

The present disclosure provides technologies for treatment and/or prevention of certain diseases, disorders, and/or conditions, and furthermore provides technologies for assessing one or more characteristics of agents useful in such treatment and/or prevention.

In some embodiments, provided technologies relate to treatment of nausea and/or vomiting, and in some embodiments relate in particular to induced (e.g., chemotherapy-induced) nausea and/or vomiting. Alternatively, or additionally, in some embodiments, provided technologies relate to treatment or prevention of one or more feeding disorders (e.g., anorexia nervosa).

Nausea and/or vomiting are experienced with a number of diseases and disorders, and furthermore, can result from therapeutic treatments, such as chemotherapy.

Regardless of the situation under which nausea and/or vomiting arise, both are generally considered unpleasant and undesirable. Therefore, subjects seek to avoid nausea and/or vomiting; such avoidance can lead, for example, to noncompliance with therapeutic treatments, e.g., when the patient perceives an association between such treatments and experienced nausea and/or vomiting.

The present disclosure appreciates that certain biological pathways involved in surveillance of and/or response to toxins (e.g., toxin detoxification pathways), and particularly certain signaling pathways that transduce detection of translational deficits into induction of detoxification genes are conserved across animal phylogeny may also suppress aberrant human xenobiotic responses; the present disclosure teaches that such agents may have therapeutic potential in the treatment of certain diseases, disorders, or conditions, specifically including, for example, nausea and/or vomiting (e.g., chemotherapy-induced nausea, a major problem in cancer therapy), and/or in feeding disorders such as anorexia nervosa.

The present disclosure thus provides model systems (e.g., C. elegans) of characterizing agents for usefulness as therapeutic agents as described herein. Moreover, the present disclosure documents use of such systems to identify and/or characterize certain useful such agents.

Among other things, the present disclosure teaches that certain carotenoid compounds (e.g., certain C50 carotenoid compounds) are useful in the treatment and/or prevention of diseases, disorders and/or conditions such as nausea and/or vomiting (e.g., induced nausea such as chemotherapy-induced nausea) and/or one or more feeding disorders (e.g., anorexia nervosa).

For example, the present disclosure provides that certain carotenoid compounds (e.g., certain C50 carotenoid compounds) inhibit detoxification pathways, including those activated in response to translational deficits induced by toxins and/or by a mutation in a translation component. The present disclosure recognizes that carotenoids are generally well-tolerated by mammals, including humans, and teaches that they are an attractive class of compounds for therapeutic use as described herein (e.g., for the treatment and/or prevention of nausea and/or vomiting, and/or of one or more feeding disorders).

The present disclosure particularly recognizes that certain C50 carotenoid compounds are naturally produced by microbes (as reviewed, for example, by Hencke et al., “C50 Carotenoids: Occurrence, Biosynthesis, Glycosylation, and Metabolic Engineering for their Overproduction”, Chapter 5 of Bio-pigmentation and Biotechnological Implementations, Ed. Singh, Wiley & Sons, 2017). In some embodiments, delivery of carotenoid compounds (e.g., C50 carotenoid compounds) for therapeutic use as described herein, may be accomplished by administration of a composition that is or comprises a microbe that produces one or more carotenoid compound(s) of interest, or an extract or purified component thereof. In some embodiments, such administration may be of a viable (e.g., microbe), and in certain embodiments may achieve colonization of administered microbe(s) in the recipient (e.g., as part of the recipient's microbiome.

Among other things, the present disclosure appreciates that embodiments involving administration of microbe(s), and particularly of viable (e.g., live) microbe(s) may offer certain advantages, such as, for example, reduced administrations (e.g., reduced frequency of dosing, term of dosing, total number of administered doses, volume and/or concentration of administered doses, and/or combinations thereof, etc), reduced costs, long term efficacy, etc.

Those skilled in the art, reading the present disclosure will appreciate, however, that delivery of carotenoid compounds (e.g., C50 carotenoid compounds) for treatment as described herein is not limited to delivery of microbe(s), or even to extracts and/or components thereof; rather, useful carotenoid compounds (e.g., as described herein) may be prepared in whole or in part by chemical synthesis and/or may be purified from microbial sources (e.g., cultured microbial cells, which may be naturally-occurring and/or genetically or otherwise engineered cells).

Those skilled in the art will further appreciate that any of a variety of delivery routes and/or forms may be utilized to administer compositions that deliver (e.g., that are or comprise microbe(s) and/or extract(s) or component(s) thereof and/or one or more pure carotenoid compound(s) as described herein) useful carotenoid compounds as described herein. In many embodiments, compositions are administered orally (e.g., via a pill, tablet, capsule, powder, lozenge, syrup, elixir, etc). In some embodiments, oral administration is via a nutritional source such as a food or drink.

One challenge associated with development of useful treatments for nausea and/or vomiting has been a lack of an animal model. The present disclosure documents that Caenorhabditis elegans can provide an effective model for nausea and/or vomiting. Among other things, the present disclosure provides that C. elegans can be useful to characterize (e.g., to screen) agents to assess their impact on and/or usefulness in treating nausea and/or vomiting. For example, microbial toxins and virulence factors often target translation machinery. C. elegans responds to translational deficits (e.g., as may result from exposure to toxins and/or from mutation of a translation component) by induction of detoxification and defense response genes. In accordance with the present disclosure, agents that inhibit this induction can be useful in treatment of nausea and/or vomiting, and assessment of such inhibition can be useful to characterizing (e.g., screening) such agents.

Still further, the present disclosure demonstrates that certain carotenoid compounds (e.g., certain C50 carotenoid compounds) can inhibit the C. elegans translation deficit surveillance and response pathways (e.g., the C. elegans xenobiotic detoxification response to a translational deficit); such carotenoid compounds may be useful in accordance with the present disclosure in therapeutic applications, such as to treat nausea and/or vomiting. For example, the present disclosure specifically documents that a C50 carotenoid compound produced by Kocuria rhizophila inhibits C. elegans translation deficit surveillance and response pathways. Among other things, the present disclosure describes a genetic analysis, that identifies the biosynthetic pathway for this carotenoid as mediating inhibition of the C. elegans translational toxin defense response. Furthermore, the present disclosure documents that K. rhizophila extracts (i) recapitulate suppression of the C. elegans xenobiotic detoxification response to a translational deficit; and (ii) restore the ability of K. rhizophila carotenoid mutants to inhibit such detoxification response to defects in translation.

Additionally, the present disclosure documents that other carotenoid compounds (e.g., C50 carotenoid compounds produced by other bacterial species) also inhibit the C. elegans translation deficit surveillance and response pathways (e.g., the C. elegans xenobiotic detoxification response to a translational deficit). For example, the present disclosure documents that C. glutamicum, which produces the produces the C50 carotenoid decaprenoxanthin, also inhibits C. elegans detoxification responses, and furthermore that C. glutamicum carotenoid biosynthesis mutants are defective in this inhibition.

Still further, the present disclosure documents that yet another bacterial species Arthrobacter arilaitensis, which also produces a C50 carotenoid (specifically, decaprenoxanthin), also inhibits C. elegans detoxification responses.

Without wishing to be bound by any particular theory, the present disclosure proposes that carotenoid compounds as described herein (e.g., C50 carotenoid compounds) suppress induction of xenobiotic detoxification by inhibiting a C. elegans bile acid signaling pathway that transduces detection of translational deficits into induction of detoxification genes. Suppression of translational surveillance by carotenoid compounds (e.g., C50 carotenoid compounds) as documented herein disables drug detoxification responses so that the potency of translational inhibitory drugs is enhanced. Thus, in some embodiments, carotenoid compounds useful in accordance with the present disclosure may be characterized by their ability to increase potency of translational inhibitory drugs. For example, in some embodiments, a useful carotenoid compound is characterized in that, when such carotenoid compound is contacted with C. elegans in the presence of a translational inhibitory drug, one or more features of that translational inhibitory drug's impact on the C. elegans is enhanced relative to that observed under otherwise comparable conditions (e.g., presence of the same translational inhibitory drug at the same concentration, etc.) absent the carotenoid compound.

The present disclosure also demonstrates that certain carotenoid compounds (e.g., certain C50 carotenoid compounds) inhibit coupling of translational surveillance to food aversion behaviors in C. elegans that are normally induced by translational inhibitory drugs. Thus, in some embodiments, carotenoid compounds useful in accordance with the present disclosure may be characterized by their impact on C. elegans food aversion behaviors in the presence of relevant toxins. For example, in some embodiments, a useful carotenoid compound may be characterized in that, when such compound is contacted with C. elegans in the presence of a toxin targeting protein translation, one or more features of that toxin's impact on C. elegans food avoidance behavior is altered relative to that observed under otherwise comparable conditions (e.g., presence of the same toxin at the same concentration, etc.) absent the carotenoid compound. In some embodiments, agents (e.g., carotenoid compounds such as C50 carotenoid compounds) shown to impact food aversion behavior as described herein may be particularly useful in treatment of one or more food aversion disorders such as, for example, anorexia nervosa.

Thus, provided herein are methods for the treatment of nausea and/or vomiting, or for the reduction of food aversion, in a subject. The methods include administering to a subject in need thereof a therapeutically effective amount of a C50 carotenoid compound. In some embodiments, the subject has or is at risk of developing nausea and/or vomiting associated with chemotherapy or radiation, e.g., chemotherapy-induced nausea and vomiting (CINV) or radiation-induced nausea and vomiting (RINV).

In some embodiments, the subject has or is at risk of developing post-operative nausea and vomiting (PONV).

In some embodiments, the C50 carotenoid compound is selected from the group consisting of decaprenoxanthin, C50-astaxanthin, C50-β-carotene, C50-carotene (n=3) (16,16-diisopentenylphytoene), C50-zeaxanthin, C50-caloxanthin, C50-nostoxanthin sarcinaxanthin, sarprenoxanthin, acyclic C50 carotenoid bacterioruberin, C50-canthaxanthin, C50-lycopene, C50-phytoene, and combinations thereof. In some embodiments, the C50 carotenoid compound is decaprenoxanthin.

In some embodiments, the step of administering comprises administering a composition that is or comprises (i) a C50-carotenoid-compound-synthesizing microbe or component thereof, (ii) an extract from a C50-carotenoid-compound-synthesizing microbe, (iii) an extracted carotenoid compound, or (iv) a combination thereof. In some embodiments, the C50-carotenoid-compound-synthesizing microbe is viable or alive. In some embodiments, the step of administering comprises administering a sufficient amount of the microbe to colonize the subject's microbiome. In some embodiments, the composition comprises or is prepared from a culture of the microbe. In some embodiments, the microbe is a strain that is found in nature. In some embodiments, the microbe is an engineered microbe. In some embodiments, the engineered microbe comprises a genetic alteration relative to an otherwise comparable reference microbe so that it produces the C50 carotenoid compound at an absolute or relative level different from that of the reference microbe.

In some embodiments, the step of administering comprises administering a composition that comprises or delivers a synthesized C50 carotenoid compound. In some embodiments, the C50-carotenoid-compound-synthesizing microbe is selected from the group consisting of Kocuria rhizophila, Corynebacterium glutamicum, Arthrobacter arilaitensis, and combinations thereof.

Also provided herein are therapeutic compositions for oral delivery comprising a therapeutically effective amount of a C50 carotenoid compound, and a pharmaceutically acceptable carrier.

In some embodiments, the composition comprises a microbe that synthesizes the C50 carotenoid compound. In some embodiments, the microbe is a cultured microbe. In some embodiments, the microbe is an engineered microbe. In some embodiments, the engineered microbe comprises a genetic alteration relative to an otherwise comparable reference microbe so that it produces the C50 carotenoid compound at an absolute or relative level different from that of the reference microbe. In some embodiments, the microbe is living or viable. In some embodiments, the microbe has been killed. In some embodiments, the microbe is selected from the group consisting of Kocuria rhizophila, Corynebacterium glutamicum, Arthrobacter arilaitensis, and combinations thereof.

In some embodiments, the C50 carotenoid compound is at least 20% w/w of the composition.

In some embodiments, the C50 carotenoid compound is purified.

In some embodiments, the C50 carotenoid compound has a chemical structure found in nature.

In some embodiments, the C50 carotenoid compound is an analog of a reference C50 carotenoid compound found in nature.

In some embodiments, the C50 carotenoid compound is selected from the group consisting of decaprenoxanthin, C50-astaxanthin, C50-β-carotene, C50-carotene (n=3) (16,16-diisopentenylphytoene), C50-zeaxanthin, C50-caloxanthin, C50-nostoxanthin sarcinaxanthin, sarprenoxanthin, acyclic C50 carotenoid bacterioruberin, C50-canthaxanthin, C50-lycopene, C50-phytoene, and combinations thereof. In some embodiments, the C50 carotenoid compound is decaprenoxanthin.

In some embodiments, the therapeutic composition is a liquid, syrup, tablet, troche, gummy, capsule, powder, gel, or film.

Further, provided herein are methods for manufacturing the therapeutic compositions. For example, the method can include the steps of combining a pharmaceutically acceptable carrier with a C50 carotenoid compound; and formulating the combination into the therapeutic composition.

In some embodiments, the step of combining comprises combining the pharmaceutically acceptable carrier with a microbe that synthesizes the C50 carotenoid compound.

In some embodiments, the step of combining comprises combining the pharmaceutically acceptable carrier with a chemically synthesized C50 carotenoid compound.

Also provided herein are methods for the treatment of nausea and/or vomiting, or for the reduction of food aversion, in a subject, comprising a step of administering to a subject in need thereof: (i) a C50-carotenoid-compound-synthesizing microbe or component thereof, (ii) a C50-carotenoid-compound-synthesizing microbe extract, or (iii) an extracted C50-carotenoid compound, or (iv) a combination thereof.

In some embodiments, the subject has or is at risk of developing nausea and/or and vomiting associated with chemotherapy or radiation.

In some embodiments, the subject has or is at risk of developing chemotherapy-induced nausea and vomiting (CINV) or radiation-induced nausea and vomiting (RINV). In some embodiments, the subject has or is at risk of developing post-operative nausea and vomiting (PONV).

In some embodiments, the C50 carotenoid compound-synthesizing microbe is selected from the group consisting of Kocuria rhizophila, Corynebacterium glutamicum, Arthrobacter arilaitensis, and combinations thereof.

Additionally, provided herein is the use of a C50 carotenoid compound for treating nausea and/or vomiting, or for reducing food aversion, in a subject in need thereof, and a C50 carotenoid compound for use in treating nausea and/or vomiting or for reducing food aversion in a subject in need thereof.

Also provided is the use of a C50-carotenoid-compound-synthesizing microbe for treating nausea and/or vomiting or for reducing food aversion, and a C50-carotenoid-compound-synthesizing microbe for use in treating nausea and/or vomiting or for reducing food aversion, in a subject in need thereof.

In some embodiments, the C50 carotenoid compound-synthesizing microbe is selected from the group consisting of Kocuria rhizophila, Corynebacterium glutamicum, Arthrobacter arilaitensis, and combinations thereof.

Also provided herein are methods for assessing a carotenoid compound for anti-nausea and/or anti-emesis activity. The methods include the steps of (i) contacting a system with the carotenoid compound; and (ii) determining whether the carotenoid compound altered a feature of the system, wherein the feature is associated with nausea and/or vomiting.

In some embodiments, the step of determining comprises comparing the feature before and after performance of the step of contacting.

In some embodiments, the step of determining comprises comparing the feature after the step of contacting with a comparable reference.

In some embodiments, the comparable reference is a historical reference.

In some embodiments, the comparable reference is a negative control reference.

In some embodiments, the comparable reference is a positive control reference.

In some embodiments, the system is or comprises C. elegans.

In some embodiments, the feature is a level of food aversion.

In some embodiments, the feature is level or activity of a nucleic acid or protein, or form thereof.

In some embodiments, the feature is or comprises an aspect of a xenobiotic detoxification response.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of embodiments of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A-E.

A) pgp-5p::gfp induction was significantly reduced in eft-3(q145); pgp-5p::gfp animals fed on K. rhizophila wildtype, while mutants in K. rhizophila crtEb(e17), K. rhizophila crtI(e10), and K. rhizophila crtYe(e2) did not suppress GFP induction.

B) K. rhizophila feeding reduced pgp-5p::gfp induction in eft-3(q145); pgp-5p::gfp animals significantly, while in K. rhizophila mutants, pgp-5p::gfp expression was not affected. Unpaired t-test, **P<0.01. Mean±s.d is shown. The number of animals analyzed per condition is shown above each bar. ns, was not significant compared to eft-3(q145); pgp-5p::gfp fed on E. coli OP50.

C) A discoloration phenotype of K. rhizophila mutants.

D) A diagrammatic representation of mutations in carotenoid cluster in the K. rhizophila.

E) A putative C50 carotenoid biosynthetic pathway in K. rhizophila.

FIGS. 2A-G

A) pgp-5p::gfp induction was significantly reduced in eft-3(q145); pgp-5p::gfp animals fed on C. glutamicum wildtype, while mutants in C. glutamicum ΔcrtEb, ΔcrtI, ΔcrtY, and ΔcrtB did not suppress GFP induction.

B) A quantification of pgp-5p::gfp expression in eft-3(q145); pgp-5p::gfp animals fed on C. glutamicum wildtype, ΔcrtEb, ΔcrtI, ΔcrtY, and ΔcrtB mutants. Unpaired t-test, ****P<0.0001. Mean±s.d is shown. The number of animals analyzed per condition is shown above each bar. ns, was not significant compared to eft-3(q145); pgp-5p::gfp fed on E. coli OP50.

C) pgp-5p::gfp induction was significantly reduced in eft-3(q145); pgp-5p::gfp animals fed on A. arilaitensis wildtype.

D) A TLC of a K. rhizophila extract showing orange pigments.

E) An HPLC of a K. rhizophila extract showing absorbance of orange pigments. The inset shows the elution times and absorbance of different peaks from the extract.

F) 750 μg/ml of a K. rhizophila extract inhibited pgp-5p::gfp in eft-3(q145); pgp-5p::gfp animals.

G) A quantification of pgp-5p::gfp expression in eft-3(q145); pgp-5p::gfp animals fed on K. rhizophila wildtype, K. rhizophila crtEb(e17), K. rhizophila crtI(e10), and K. rhizophila crtEb(e6) containing either a control extract or an K. rhizophila extract.

FIGS. 3A-F

A) pgp-5p::gfp induction in response to 10 mg/ml of hygromycin was significantly reduced in animals fed on K. rhizophila wildtype, while mutants in K. rhizophila crtEb(e17), or K. rhizophila crtI(e10) did not suppress the GFP induction.

B) Animals treated with a K. rhizophila carotenoid extract were hypersensitive to hygromycin. Unpaired t-test, ****P<0.0001 compared to the wildtype worms fed on E. coli OP50 containing solvent extract and hygromycin. Mean±s.d is shown. Data were collected from three independent trials of at least 20 animals for each condition. ns, was not significant compared to wildtype fed on E. coli OP50 containing solvent extract with no hygromycin.

C) Animals treated with a K. rhizophila carotenoid extract were hypersensitive to emetine.

D) Animals treated with a K. rhizophila carotenoid extract were hypersensitive to cisplatin.

E) Animals treated with a K. rhizophila carotenoid extract failed to avoid hygromycin compared animals treated with a control solvent and hygromycin.

F) Animals treated with a K. rhizophila carotenoid extract failed to avoid cisplatin-compared animals treated with a control solvent and cisplatin. Unpaired t-test, ****P<0.01, **P<0.0001. Mean±s.d is shown. ns, was not significant

FIGS. 4A-E

A) pgp-5p::gfp was constitutively induced in animals fed on E. coli OP50 or K. rhizophila expressing ZIP-2::mCherry in the intestine under the control of vha-6 promoter.

B) Supplementation of bile acids suppressed a K. rhizophila-induced pgp-5p::gfp activation defect in eft-3(q145); pgp-5p::gfp animals.

C) A quantification of the suppression of a K. rhizophila-induced pgp-5p::gfp activation defect in eft-3(q145); pgp-5p::gfp animals by bile acids shown in FIG. 4B. Unpaired t-test, ***P<0.0001. Mean±s.d is shown. The number of animals analyzed per condition is shown above each bar. ns, was not significant compared to eft-3(q145); pgp-5p::gfp fed on E. coli OP50.

D) lbp-5 RNAi, chc-1RNAi, fcho-1 RNAi, and dyn-1 RNAi suppressed a K. rhizophila-induced pgp-5p::gfp activation defect in eft-3(q145); pgp-5p::gfp animals while rme-1 RNAi or rab-5 RNAi did not suppress the GFP induction.

E) A working model of how K. rhizophila carotenoid extract may suppress the induction of xenobiotic detoxification response.

FIGS. 5A-G

A) K. rhizophila feeding inhibited induction of pgp-5p::gfp in eft-3(q145); pgp-5p::gfp animals, while in K. rhizophila mutants, pgp-5p::gfp expression was not affected.

B) K. rhizophila feeding suppressed pgp-5p::gfp induction in eft-3(q145); pgp-5p::gfp animals within 12 hours of feeding.

C) While pgp-5p::gfp animals fed on vrs-2 dsRNA and transferred to E. coli showed induction of gfp, in animals transferred to K. rhizophila plates, pgp-5p::gfp expression was reduced.

D) While pgp-5p::gfp animals fed on rpl-1 dsRNA and transferred to E. coli showed induction of gfp, in animals transferred to K. rhizophila plates, pgp-5p::gfp expression was reduced.

E) K. rhizophila feeding reduced pgp-5p::gfp induction in eft-3(q145); pgp-5p::gfp animals significantly, while in K. rhizophila mutants, the pgp-5p::gfp expression was not affected.

F) Colony color of K. rhizophila wildtype was different from the colony color of six mutants.

G) Colony color of K. rhizophila mutants observed from a EMS screen.

FIGS. 6A-C

A) K. rhizophila mutants failed to suppress induction of pgp-5p::gfp in eft-3(q145); pgp-5p::gfp animals.

B) A discoloration phenotype observed from 23 genome sequenced mutants.

C) A discoloration phenotype of observed from C. glutamicum wildtype, ΔcrtEb, ΔcrtI, ΔcrtY, and ΔcrtB mutants.

FIG. 7

An operon structure of bacteria that contain putative gene cluster that might produce decaprenoxanthin.

FIG. 8

An alignment of CrtI protein in different genera. Amino acid conservation across sequences found in different genera is shown.

FIG. 9

An alignment of CrtB protein in different genera. Amino acid conservation across sequences found in different genera is shown.

FIG. 10

An alignment of CrtEb protein in different genera. Amino acid conservation across sequences found in different genera is shown.

FIG. 11

One exemplary biochemical isolation of decaprenoxanthin containing extract from K. rhizophila.

FIGS. 12A-B-C

A spectrophotometric analysis of methanolic extracts from K. rhizophila wildtype, crtI(e10) and crtb(e6) (12A); wildtype, crtEb(e17) and crtYf(e18) (12B); and wildtype, crtEb(e17) and crtYe(e22) (12C).

FIGS. 13A-E

A) K. rhizophila did not induce hsp-4p::gfp.

B) K. rhizophila wildtype as well as crtEb(e17) or crtI(e10) mutants did not induce hsp-6p::gfp.

C) K. rhizophila wildtype, as well as crtEb(e17), crtI(e10), crtYf(e2) or crtYe(e22) mutants, induced clec-60p::gfp.

D) K. rhizophila wildtype, as well as crtEb(e17) or crtI(e10) mutants, did not induce F35E12.5p::gfp.

E) K. rhizophila feeding induced suppression of pgp-5p::gf in eft-3(q145); pgp-5p::gfp animals was reversible.

FIGS. 14A-D.

A) N-acetyl cysteine, ascorbic acid, trolox or Resveratrol did not suppress pgp-5p::gfp in eft-3(q145); pgp-5p::gfp animals.

B) beta-carotene or Astaxanthin did not suppress pgp-5p::gfp in eft-3(q145); pgp-5p::gfp animals.

C) E. coli expressing either zeaxanthin, neurosporene, violaxanthin, delta-carotene, or alpha-carotene did not suppress pgp-5p::gfp in eft-3(q145); pgp-5p::gfp animals.

D) pgp-5p::gfp induction in response to 10 μg/ml and 20 g/ml of hygromycin was significantly reduced in animals fed on K. rhizophila wildtype. pgp-5p::gfp induction in response to 50 mg/ml hygromycin in normal in both animals fed on E. coli OP50 as well as on K. rhizophila wildtype.

FIGS. 15A-E

A) pgp-5p::gfp induction in response to 2.5 g/ml or 6.25 g/ml of emetine was significantly reduced in animals fed on K. rhizophila wildtype. However, in animals treated with 12.5 g/ml of emetine, pgp-5p::gfp induction in response was partially reduced in animals fed on K. rhizophila. In animals treated with 25 g/ml of emetine, induction of pgp-5p::gfp was normal both animals treated with either E. coli OP50 or K. rhizophila wildtype.

B) pgp-5p::gfp induction in response to 6.25 μg/ml of emetine was significantly reduced in animals fed on K. rhizophila wildtype, while mutants in K. rhizophila crtEb(e17) or K. rhizophila crtI(e10) did not suppress the GFP induction.

C) pgp-5p::gfp induction in response to 1 mM of cisplatin was significantly reduced in animals fed on K. rhizophila wildtype, while mutants in K. rhizophila crtEb(e17), crtYe(e22) or crtI(e10) did not suppress the GFP induction.

D) Carotenoids by themselves were not toxic to the worms in the absence of hygromycin.

E) Animals fed on control extract or K. rhizophila extract were sensitive to Antimycin.

FIGS. 16A-C

A) ZK892.4 and C24A3.4 proteins share sequence similarity.

B) Inactivation of ZK892.4 RNAi or C24A3.4 RNAi did not suppress pgp-5p::gfp induction in eft-3(q145); pgp-5p::gfp animals, while double RNAi of ZK892.4 and C24A3.4 suppressed the gfp induction.

C) RNAi of chc-1, fcho-1, or lbp-5 did not induce pgp-5p::gfp.

DEFINITIONS

Administration: As used herein, the term “administration” typically refers to the administration of a composition to a subject or system to achieve delivery of an agent to the subject or system. In some embodiments, the agent is, or is included in, the composition; in some embodiments, the agent is generated through metabolism of the composition or one or more components thereof. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some particular embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e.g. intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc. In many embodiments provided by the present disclosure, administration is oral administration. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

Analog: As used herein, the term “analog” refers to a substance that shares one or more particular structural features, elements, components, or moieties with a reference substance. Typically, an “analog” shows significant structural similarity with the reference substance, for example sharing a core or consensus structure, but also differs in certain discrete ways. In some embodiments, an analog is a substance that can be generated from the reference substance, e.g., by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be generated through performance of a synthetic process substantially similar to (e.g., sharing a plurality of steps with) one that generates the reference substance. In some embodiments, an analog is or can be generated through performance of a synthetic process different from that used to generate the reference substance.

Carotenoid compound: As used herein, the term “carotenoid compound” refers to compounds that are members of the structurally diverse class of naturally-occurring carotenoid pigments, and structural analogs thereof. In nature, carotenoid compounds are typically synthesized from isoprenoid pathway intermediates. Carotenoids can be acyclic or cyclic, and may or may not contain oxygen, so that the term “carotenoids”, in some embodiments, can include both carotenes and xanthophylls. Many carotenoids have strong light absorbing properties. In general, carotenoids are hydrocarbon compounds having a conjugated polyene carbon skeleton formally derived from the five-carbon compound isopentenyl pyrophosphate. In some embodiments, carotenoid compounds may be triterpenes (C30 diapocarotenoids), tetraterpenes (C40 carotenoids), or other compounds that are, for example, C35, C50, C60, C70, C80 in length or other lengths. In some embodiments, a carotenoid may have a length in excess of C200. More than 1000 different carotenoids have been identified in nature. Carotenoids include but are not limited to: antheraxanthin, adonirubin, adonixanthin, astaxanthin, canthaxanthin, capsorubrin, β-cryptoxanthin, α-carotene, β-carotene, β, ψ-carotene, δ-carotene, ε-carotene, echinenone, 3-hydroxyechinenone, 3′-hydroxyechinenone, γ-carotene, ψ-carotene, 4-keto-γ-carotene, ζ-carotene, α-cryptoxanthin, deoxyflexixanthin, diatoxanthin, 7,8-didehydroastaxanthin, didehydrolycopene, fucoxanthin, fucoxanthinol, isorenieratene, β-isorenieratene, lactucaxanthin, lutein, lycopene, myxobactone, neoxanthin, neurosporene, hydroxyneurosporene, peridinin, phytoene, rhodopin, rhodopin glucoside, 4-keto-rubixanthin, siphonaxanthin, spheroidene, spheroidenone, spirilloxanthin, torulene, 4-keto-torulene, 3-hydroxy-4-keto-torulene, uriolide, uriolide acetate, violaxanthin, zeaxanthin-β-diglucoside, zeaxanthin, and C30 carotenoids. Additionally, carotenoid compounds include derivatives of these molecules, which may include hydroxy-, methoxy-, oxo-, epoxy-, carboxy-, or aldehydic functional groups. For example, carotenoids include oxygenated derivatives. Further, included carotenoid compounds include ester (e.g., glycoside ester, fatty acid ester) and sulfate derivatives (e.g., esterified xanthophylls).

Carotenogenic modification: The term “carotenogenic modification”, as used herein, refers to a modification of a host organism that adjusts production of one or more carotenoids, as described herein. For example, a carotenogenic modification may increase the production level of one or more carotenoids, and/or may alter relative production levels of different carotenoids. In principle, an inventive carotenogenic modification may be any chemical, physiological, genetic, or other modification that appropriately alters production of one or more carotenoids in a host organism produced by that organism as compared with the level produced in an otherwise identical organism not subject to the same modification. In most embodiments, however, the carotenogenic modification will comprise a genetic modification, typically resulting in increased production of one or more selected carotenoids. In some embodiments, the selected carotenoid is one or more C50 carotenoid compounds.

Carotenoid biosynthesis polypeptide: The term “carotenoid biosynthesis polypeptide” refers to any polypeptide that is involved in the synthesis of one or more carotenoids. To mention but a few, these carotenoid biosynthesis polypeptides include, for example, polypeptides of phytoene synthase, phytoene dehydrogenase (or desaturase), lycopene cyclase, carotenoid ketolase, carotenoid hydroxylase, astaxanthin synthase, carotenoid epsilon hydroxylase, lycopene cyclase (beta and epsilon subunits), carotenoid glucosyltransferase, and acyl CoA:diacyglycerol acyltransferase.

Carotenoid-Compound-Synthesizing Microbe: As used herein, the phrase “carotenoid-compound-synthesizing microbe” refers to a microbe (e.g., algae, fungi, bacteria) that synthesizes one or more carotenoid compounds. In some embodiments, a carotenoid-compound-synthesizing microbe may naturally synthesize one or more carotenoids compounds. In some embodiments, a carotenoid-compound-synthesizing microbe includes a carotenogenic modification. In some embodiments, a carotenoid-compound-synthesizing compound may be genetically modified (e.g., to have one or more genetic alterations) so that it synthesizes one or more carotenoids at an absolute or relative level different from that of an otherwise comparable reference microbe that has not been so genetically modified (i.e., does not contain the genetic alteration(s)). For example, in some embodiments, a carotenoid-compound-synthesizing microbe has been genetically engineered to synthesize at least one carotenoid compound not synthesized by the microbe absent the genetic engineering. Alternatively, in some embodiments, a carotenoid-compound-synthesizing microbe may have been genetically engineered so that its synthesis of one or more particular carotenoid compounds may be at a higher level relative to the microbe absent the genetic engineering. In some embodiments, a higher level may be assessed in reference to a threshold level; in some embodiments, a higher level may be assessed in reference to another compound (e.g., another carotenoid compound) also produced by the microbe (prior to the genetic engineering). In some particular embodiments, a carotenoid-compound-synthesizing microbe may have been genetically modified to add or increase expression of one or more genes encoding a carotenoid biosynthesis polypeptide. Alternatively, or additionally, in some embodiments, a carotenoid-compound-synthesizing microbe may have been genetically modified to increase carbon flow through a carotenoid biosynthesis pathway (e.g., by reducing carbon diversion into one or more other biosynthesis or metabolic pathways). In some embodiments, a carotenoid-compound-synthesizing microbe may synthesize one or more carotenoid compounds having a particular number of carbon units. For example, in some embodiments, a carotenoid-compound-synthesizing microbe may synthesize (naturally or as a result of genetic modifications) one or more C50 carotenoid compounds; such a microbe may be referred to as a C50-synthesizing microbe.

Comparable: As used herein, the term “comparable” refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison therebetween so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

Dosage form: Those skilled in the art will appreciate that the term “dosage form” may be used to refer to a physically discrete unit of an agent (e.g., a therapeutic agent) for administration to a subject. Typically, each such unit contains a predetermined quantity of agent. In some embodiments, such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen). Those of ordinary skill in the art appreciate that the total amount of a therapeutic composition or agent administered to a particular subject is determined by one or more attending physicians and may involve administration of multiple dosage forms.

Dosing regimen: Those skilled in the art will appreciate that the term “dosing regimen” may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which is separated in time from other doses. In some embodiments, individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population.

Engineered: In general, the term “engineered” refers to the aspect of having been manipulated by the hand of man. For example, a cell or organism is considered to be “engineered” if it has been manipulated so that its genetic information is altered (e.g., new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols). As is common practice and is understood by those in the art, progeny of an engineered polynucleotide or cell are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity.

Excipient: as used herein, refers to an inactive (e.g., non-therapeutic) agent that may be included in a pharmaceutical composition, for example to provide or contribute to a desired consistency or stabilizing effect. In some embodiments, suitable pharmaceutical excipients may include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

Functional: As used herein, a “functional” biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized. A biological molecule may have two functions (i.e., bifunctional) or many functions (i.e., multifunctional).

Improve, increase, inhibit or reduce: As used herein, the terms “improve”, “increase”, “inhibit”, “reduce”, or grammatical equivalents thereof, indicate values that are relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single individual) under otherwise comparable conditions absent presence of (e.g., prior to and/or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent. In some embodiments, an appropriate reference measurement may be or comprise a measurement in comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment. In some embodiments, an appropriate reference is a negative reference; in some embodiments, an appropriate reference is a positive reference.

Isolated: as used herein, refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) designed, produced, prepared, and/or manufactured by the hand of man. In some embodiments, an isolated substance or entity may be enriched; in some embodiments, an isolated substance or entity may be pure. In some embodiments, isolated substances and/or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated. In some embodiments, isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is “pure” if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may still be considered “enriched”, “isolated” or even “pure”, after having been combined with certain other components such as, for example, one or more carriers or excipients (e.g., buffer, solvent, water, etc.); in such embodiments, percent isolation or purity of the substance is calculated without including such carriers or excipients. Those skilled in the art are aware of a variety of technologies for isolating (e.g., enriching or purifying) substances or agents (e.g., using one or more of fractionation, extraction, precipitation, or other separation).

Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, a pharmaceutical composition may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue, capsules, powders, etc. In some embodiments, an active agent may be or comprise a cell or population of cells (e.g., a culture, for example of a carotenoid-compound-synthesizing microbe); in some embodiments, an active agent may be or comprise an extract or component of a cell or population (e.g., culture) of cells. In some embodiments, an active agent may be or comprise an isolated, purified, or pure compound. In some embodiments, an active agent may have been synthesized in vitro (e.g., via chemical and/or enzymatic synthesis). In some embodiments, an active agent may be or comprise a natural product (whether isolated from its natural source or synthesized in vitro).

Pharmaceutically acceptable: As used herein, the term “pharmaceutically acceptable” which, for example, may be used in reference to a carrier, diluent, or excipient used to formulate a pharmaceutical composition as disclosed herein, means that the carrier, diluent, or excipient is compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

Pharmaceutically acceptable carrier: As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be is “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

Prevention: The term “prevention”, as used herein, refers to a delay of onset, and/or reduction in frequency and/or severity of one or more symptoms of a particular disease, disorder or condition. In some embodiments, prevention is assessed on a population basis such that an agent is considered to “prevent” a particular disease, disorder or condition if a statistically significant decrease in the development, frequency, and/or intensity of one or more symptoms of the disease, disorder or condition is observed in a population susceptible to the disease, disorder, or condition. In some embodiments, prevention may be considered complete, for example, when onset of a disease, disorder or condition has been delayed for a predefined period of time.

Reference: As used herein describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control. In some embodiments, a reference is a negative control reference; in some embodiments, a reference is a positive control reference.

Risk: as will be understood from context, “risk” of a disease, disorder, and/or condition refers to a likelihood that a particular individual will develop the disease, disorder, and/or condition. In some embodiments, risk is expressed as a percentage. In some embodiments, risk is from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 up to 100%. In some embodiments risk is expressed as a risk relative to a risk associated with a reference sample or group of reference samples. In some embodiments, a reference sample or group of reference samples have a known risk of a disease, disorder, condition and/or event. In some embodiments a reference sample or group of reference samples are from individuals comparable to a particular individual. In some embodiments, relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

Sample: As used herein, the term “sample” typically refers to an aliquot of material obtained or derived from a source of interest. In some embodiments, a source of interest is a biological or environmental source. In some embodiments, a source of interest may be or comprise a cell or an organism, such as a microbe, a plant, or an animal (e.g., a human). In some embodiments, a source of interest is or comprises biological tissue or fluid. In some embodiments, a biological tissue or fluid may be or comprise amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle, chime, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humour, vomit, and/or combinations or component(s) thereof. In some embodiments, a biological fluid may be or comprise an intracellular fluid, an extracellular fluid, an intravascular fluid (blood plasma), an interstitial fluid, a lymphatic fluid, and/or a transcellular fluid. In some embodiments, a biological fluid may be or comprise a plant exudate. In some embodiments, a biological tissue or sample may be obtained, for example, by aspirate, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, washing or lavage (e.g., bronchioalveolar, ductal, nasal, ocular, oral, uterine, vaginal, or other washing or lavage). In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, a sample is a “primary sample” obtained directly from a source of interest by any appropriate means. In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to one or more techniques such as amplification or reverse transcription of nucleic acid, isolation and/or purification of certain components, etc.

Subject: As used herein, the term “subject” refers to an individual to which a provided treatment is administered. In some embodiments, a subject is mammal, e.g., a mammal that experiences or is susceptible to a disease, disorder, or condition as described herein; in some embodiments, a subject is a human or non-human veterinary subject, e.g., an ape, cat dog, monkey, or pig. In some embodiments, a subject is a human. In some embodiments, a patient is suffering from or susceptible to one or more diseases, disorders or conditions as described herein. In some embodiments, a patient displays one or more symptoms of a one or more diseases, disorders or conditions as described herein. In some embodiments, a patient has been diagnosed with one or more diseases, disorders or conditions as described herein. In some embodiments, the disorder or condition is or includes nausea and/or vomiting, and/or one or more food aversion disorders. In some embodiments, a subject is suffering from or susceptible to cancer, or presence of one or more tumors. In some embodiments, the subject is receiving or has received certain therapy to diagnose and/or to treat a disease, disorder, or condition. In some embodiments, a subject has received a therapy (e.g., chemotherapy, radiation, and/or surgery) that induces nausea and/or vomiting.

Symptoms are reduced: According to the present invention, “symptoms are reduced” when one or more symptoms of a particular disease, disorder or condition is reduced in magnitude (e.g., intensity, severity, etc.) and/or frequency. For purposes of clarity, a delay in the onset of a particular symptom is considered one form of reducing the frequency of that symptom.

Therapeutic regimen: A “therapeutic regimen”, as that term is used herein, refers to a dosing regimen whose administration across a relevant population may be correlated with a desired or beneficial therapeutic outcome.

Therapeutically effective amount: As used herein, is meant an amount that produces the desired effect for which it is administered. In some embodiments, the term refers to an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and/or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and/or severity of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition. Those of ordinary skill in the art will appreciate that the term “therapeutically effective amount” does not in fact require successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment. In some embodiments, reference to a therapeutically effective amount may be a reference to an amount as measured in one or more specific tissues (e.g., a tissue affected by the disease, disorder or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those of ordinary skill in the art will appreciate that, in some embodiments, a therapeutically effective amount of a particular agent or therapy may be formulated and/or administered in a single dose. In some embodiments, a therapeutically effective agent may be formulated and/or administered in a plurality of doses, for example, as part of a dosing regimen.

Treatment: As used herein, the term “treatment” (also “treat” or “treating”) refers to any administration of a therapy that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition. In some embodiments, such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition. Alternatively, or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and/or condition.

Detailed Description of Certain Embodiments

The nematode Caenorhabditis elegans surveils for deficits in essential cellular activities caused, for example, by microbial toxins, and responds by activating a cellular surveillance-activated detoxification and defenses (cSADD) response (Melo and Ruvkun, 2012). The abundant RNAs and proteins of the ribosome and other proteins that mediate the translation of mRNA to proteins can be targets of microbial toxins and virulence factors. The activity of these translational components is monitored, and decrements in protein synthesis caused by toxins or mutations can be detected. Detection of decrements is coupled via a signal transduction cascade to activate xenobiotic detoxification and behavioral responses, such as food aversion via p38MAPK, bZIP/ZIP-2 transcription factor and bile acid like biosynthetic pathways (Melo and Ruvkun, 2012; Dunbar et al., 2012; Govindan et al., 2015). By monitoring decrements in core cellular functions rather than the molecular structure of an unknown toxin, C. elegans can detect unanticipated pathogens and toxins. Many components of this signaling cascade, for example, MAP kinase and bile acid biosynthetic pathway, are conserved across animals; the present disclosure appreciates that a cSADD system of toxin surveillance and response may apply to animals other than C. elegans, and furthermore teaches that C. elegans may be useful as a model system to characterize agents that modulate this system and may be useful in certain therapeutic applications as described herein.

The present disclosure further appreciates that an animal defensive strategy analogous or homologous to the C. elegans cSADD system is likely to drive evolution of bacterial countermeasures to thwart these defense responses; there are many examples of such evolutionary arms races between host and pathogen. The present disclosure also appreciates that commensal bacteria may also seek to silence such animal defense responses to establish a benign or symbiotic relationship.

The present disclosure further appreciates that microbes (e.g., bacteria) synthesize various chemical compounds that have significant biological activities, including compounds that target the ribosome and/or associated translation factors (Berdy et al., 2005); such microbes have proven to be productive sources of drug or candidate drug compounds. The present disclosure (i) teaches useful therapeutic applications of agents that can inhibit certain detoxification pathways; (ii) provides a system for assessing one or more characteristics of agents relative to inhibition of such detoxification pathways; and (iii) identifies potential sources of such agents (e.g., microbes such as bacteria, which in some embodiments may be commensal microbes).

For example, the present disclosure provides that certain bacterial strains (e.g., those that produce certain carotenoid compounds, and in particular certain C50 carotenoid compounds) can be good sources of agents (e.g., certain useful carotenoid compounds) useful in accordance with the present disclosure. Furthermore, the present disclosure provides technologies for assessing one or more relevant characteristics of such agents.

In a microbial suppression screen of diverse bacteria for activities that abrogate activation of xenobiotic detoxification genes in C. elegans with a genetically-induced deficit in translation, a potent activity was identified from wild type K. rhizophila (Govindan et al., 2015). Genetic analysis of K. rhizophila was used to demonstrate that C50 carotenoid compounds can suppress the C. elegans translational toxin defense response.

As shown herein, K. rhizophila, which produces the C50 carotenoid compound decaprenoxanthin, inhibited the C. elegans cSADD translational and DNA damage toxin defense response. Mutants in the K. rhizophila C50 carotenoid biosynthetic pathway failed to inhibit this C. elegans xenobiotic detoxification response (FIG. 1A-C). Extracts of K. rhizophila suppressed this C. elegans xenobiotic detoxification response and also restored the ability of K. rhizophila carotenoid mutants to achieve such suppression (FIGS. 2B&C). K. rhizophila extracts also suppressed induction of C. elegans xenobiotic detoxification pathways in response to toxins that target translation (FIG. 3A, FIG. 15C). K. rhizophila C50 carotenoid compound(s) also suppressed responses to RNA interference inactivation of other genes, such as a core ribosomal protein or aminocyl tRNA synthetase, that are required for protein synthesis. A C50 carotenoid from C. glutamicum also inhibited the induction of C. elegans xenobiotic detoxification pathways in response to translation deficit, showing that C50 carotenoid regulation of the C. elegans surveillance was not limited to one particular strain of bacteria.

The present disclosure teaches that certain carotenoid compounds (e.g., certain C50 carotenoid compounds) are useful in various therapeutic applications, in particular including treatment of nausea and/or vomiting (specifically including induced nausea and/or vomiting such as chemotherapy-induced nausea and/or vomiting). Carotenoids have been most studied in photosynthetic bacteria and plants in which carotenoids are auxiliary light absorbing components in photosynthesis (Edge et al., 1997). In photosynthetic chlorophyll clusters, carotenoids can absorb and transduce light energy to photosynthetic electron transport systems, which then generate a pH gradient as reduction potential energy of photon-pumped electrons causes physical movements of multiple iron sulfur and heme proteins to move protons across the lipid bilayer. What is common among photosynthetic bacteria is a strong absorbance in the visible light wavelengths and lipid solubility of these pigments. These bacteria are highly colored because carotenoids are highly abundant and have conjugated double bond systems that delocalize electrons to extraordinarily large resonance-stabilized potential wells, with orbital transitions at much lower energy than standard biochemical bonds. Carotenoids are also known antioxidants in photosynthesis. As shown herein, however, the antioxidant activity of carotenoids does not explain the suppression of animal drug detoxification responses because other antioxidants could not suppress C. elegans surveillance.

C50 carotenoid compounds may affect animal surveillance of translational components by a simple change in membrane fluidity. As shown herein, C50 carotenoid compounds suppressed induction of the C. elegans xenobiotic detoxification response by inhibiting a bile acid biosynthetic pathway. Although bile acids were traditionally thought to be emulsifiers of fat aiding in digestion, several recent studies have found bile acids to be signaling molecules that are important in metabolism as well as immune pathways. Bile acid signaling may be involved in CINV or induction of drug detoxification response in humans. The lipid solubility and abundance of carotenoids may contribute to their anti-bile acid signaling function in C. elegans surveillance of translational and DNA damage response.

A cSADD response may not only induce xenobiotic detoxification but also food aversion behaviors. Aversion to food is an appropriate animal response because many toxins originate from bacterial pathogens that can be associated with or cause the rotting of food. Aversion to foods that are associated with the induction of xenobiotic detoxification and bacterial immunity pathways is likely to be an animal program derived from this evolutionary history. The present disclosure provides an insight that the Chemotherapy-induced Nausea and Vomiting (CINV) response in humans may be related to these xenobiotic aversion programs. Interestingly, cisplatin, which is used to block DNA replication in cancer patients, has high emetogenic potential. Emetine, which is an antibiotic that targets eukaryotic protein synthesis, is also highly emetogenic, as the name itself implies. These two drugs were not only able to induce xenobiotic detoxification response in C. elegans but also induced a strong food aversion. The pgp-5 ABC transporter gene was strongly induced by toxins that cause DNA damage, and also by the chemically very distinct toxins that inhibit translation (but not by a wide range of toxins that target other pathways such as the mitochondria or ER) (Govindan et al., 2015). Thus, the present disclosure teaches that C. elegans food aversion is a good model for studying mechanisms underlying human CINV.

Serotonergic pathways are involved in both C. elegans food aversion as well as human CINV (Melo and Ruvkun, 2012). Liver drug detoxification and elimination are a key concern in cancer chemotherapy; amplification of ABC transporters, which eliminate drugs faster, are frequently observed in drug resistant cancer patients. A K. rhizophila carotenoid extract caused hypersensitivity to xenobiotics that target protein translation, and also to xenobiotics that cause DNA damage by suppressing induction of drug detoxification pathways. Also, K. rhizophila C50 carotenoid compounds suppressed the food aversion induced by emetogenic toxins, emetine or cisplatin.

C50 carotenoid compounds, e.g., decaprenoxanthin, C50-astaxanthin, C50-β-carotene, C50-carotene (n=3) (16,16-diisopentenylphytoene), C50-zeaxanthin, C50-caloxanthin, C50-nostoxanthin sarcinaxanthin, sarprenoxanthin, acyclic C50 carotenoid bacterioruberin, C50-canthaxanthin, C50-lycopene, C50-phytoene, can be used in accordance with the present disclosure as a therapy for treating and/or reducing risk of nausea and/or vomiting, e.g., induced nausea and/or vomiting such as CINV, RINV, etc. Alternatively, or additionally, the present disclosure provides that such C50 carotenoid compounds may be useful as a therapy for treating and/or reducing risk of one or more food aversion disorders (e.g., anorexia nervosa). Still further, the present disclosure provides systems for assessing one or more characteristics of agents relevant to usefulness as a therapy as described herein (i.e., for treating and/or reducing risk of nausea and/or vomiting, and/or one or more food aversion disorders), and also demonstrates that useful such agents include compounds produced by certain microbes (e.g., bacteria), including certain commensal microbes. Using such systems, the present disclosure documents usefulness of certain carotenoid compounds (e.g., C50 carotenoid compounds), including those produced by various bacterial strains (e.g., synthesized by bacterial enzymes). Those skilled in the art, reading the present disclosure, will appreciate that various other compounds (including various other carotenoid compounds) are produced by microbes and/or can be (and/or have been) manufactured by chemical synthesis and assessed for activities such as those embodied in systems exemplified therein. Those of ordinary skill in the art, reading the present disclosure, therefore appreciate that the provides a variety of chemical agents, specifically including carotenoid compounds and exemplified by C50 carotenoids, that are amenable to assessment as described herein and/or are useful as therapeutic agents as described herein.

Methods of Treatment

Methods provided by the present disclosure include methods for the treatment of certain diseases, disorders and conditions. In some embodiments, relevant diseases, disorders and conditions may be or include nausea and/or vomiting, and/or certain food aversion disorders. In some embodiments, nausea and/or vomiting that can be treated as described herein may be associated with one or more of motion sickness, sea sickness, pregnancy (e.g., morning sickness or hyperemesis gravidarum), pain, emotional stress, gallbladder disease, heart attack, concussion or brain injury (e.g., brain tumor), overeating, gallbladder disease, infection, ulcers, gastroparesis, bowel obstruction, appendicitis, infection (e.g., viral infection) etc.

In some embodiments, nausea and/or vomiting that can be treated as described herein may be induced nausea and/or vomiting, e.g., as induced by ingestion or other exposure to toxins (e.g., food poisoning, drug-induced nausea and/or vomiting, drunkenness, etc). In some embodiments, induced nausea and/or vomiting that can be treated as described herein may be nausea and/or vomiting associated with chemotherapy or radiation. In some embodiments, induced nausea and/or vomiting that can be treated as described herein may be CINV or radiation-induced nausea and vomiting (RINV); at least three types of emesis are commonly associated with the use of chemotherapeutic agents, i.e. acute emesis, delayed emesis and anticipatory emesis. In some embodiments, induced nausea and/or vomiting that can be treated as described herein may be or include, e.g., post-operative nausea and vomiting (PONV).

Generally, methods of treatment provided by the present disclosure involve administering a therapeutically effective amount of a carotenoid compound as described herein to a subject who is in need of, or who has been determined to be in need of, such treatment.

In some embodiments, methods of treatment provided herein are prophylactic or preventative, e.g., may be administered to subjects prior to display of significant symptoms and/or to exposure to a particular expected inducement (e.g., to chemotherapy, radiotherapy, surgery, or other treatment (e.g., pharmacological treatment) that is associated with nausea and/or vomiting.

In some embodiments, methods of treatment provided herein are therapeutic, e.g., may be administered to subjects after development of significant symptoms of nausea and/or vomiting (e.g., during or after at least one episode of nausea or vomiting.

In preferred embodiments, provided methods of treatment are administered to a subject that is a mammal, e.g., a mammal that experiences a disease, disorder, or condition as described herein; in some embodiments, a subject is a human or non-human veterinary subject, e.g., an ape, cat dog, monkey, or pig.

In many embodiments, “treatment” involves ameliorating at least one symptom of a disease, disorder, or condition associated with nausea. Often, nausea results in food aversion, loss of appetite and/or reduced caloric intake, and potentially weight loss; in some embodiments, administration of a therapeutically effective amount of a carotenoid compound described herein can result in a reduction in nausea, vomiting, and/or food aversion. Alternatively or additionally, in some embodiments, administration of a therapeutically effective amount of a carotenoid compound as described herein may achieve a restoration of appetite and/or a return or approach to normal caloric intake, a reduction, cessation, or slowing of weight loss, an increase in weight/weight gain, and/or a reduction in the frequency, duration, or severity of present or future episodes of nausea, vomiting, food aversion, and/or loss of appetite.

In some embodiments, the methods can include administration of a therapeutically effective amount of a carotenoid compound before, during (e.g., concurrently with), or after administration of a treatment that is expected to be associated with nausea and/or vomiting, e.g., a subject who is about to undergo chemotherapy, radiotherapy or other treatment that is associated with nausea and vomiting.

In some embodiments, subjects who receive treatment as described herein may be receiving and/or may have received other treatment (e.g., chemotherapeutic, radiotherapeutic, surgical, etc), for example that may induce vomiting or that may be intended to treat one or more symptoms or features of a disease disorder or condition as described herein, so that provide carotenoid therapy is administered in combination with such other therapy to treat the relevant disease, disorder, or condition.

Carotenoid Compositions

Among other things, the present disclosure provides compositions that comprise or otherwise deliver carotenoid compound(s) (e.g., C50 carotenoid compounds) to subjects suffering from or susceptible to one or more diseases, disorders, or conditions as described herein.

Those skilled in the art are aware that more than 750 carotenoid compounds have been previously identified. Carotenoids are generally classified by number of 5-carbon isoprenoid units, resulting in different lengths of their carbon backbone. See, e.g., Henke et al., (2017). C50 Carotenoids: Occurrence, Biosynthesis, Glycosylation, and Metabolic Engineering for their Overproduction. In, Bio-pigmentation and Biotechnological Implementations, pp. 107-126. doi:10.1002/9781119166191.ch5; Fernandes, Introductory Chapter: Carotenoids—A Brief Overview on Its Structure, Biosynthesis, Synthesis, and Applications. In Progress in Carotenoid Research. (2018) doi:10.5772/intechopen.79542; Mezzomo et al., (2016) Carotenoids Functionality, Sources, and Processing by Supercritical Technology: A Review in J. Chemistry Volume 2016:1.

In some embodiments, carotenoid compounds useful in accordance with the present disclosure have a relatively long isoprene backbone, for example having a length within a range of about 45 to about 60 carbon atoms. In some embodiments, useful carotenoid compounds as described herein may have an isoprene backbone that is 50 carbons long, i.e., may be C50 carotenoid compounds, e.g., decaprenoxanthin, or other C50 carotenoids, e.g., C50-astaxanthin (also called decaprenoastaxanthin; see Milon et al., Helv. Chim. Acta 69, 12-24 (1986); Furubayashi et al., Nat Commun. 2015; 6: 7534) or C50-β-carotene (also called decapreno-β-carotene) (see, e.g., Karrer et al., Helv. Chim. Acta 34, 28-33 (1951); Furubayashi et al., Nat Commun. 2015; 6: 7534); 16,16′-diisopentenylphytoene (Umeno et al., J Bacteriol, 186, 1531-1536 (2004)); C50-carotene (n=3) (16,16-diisopentenylphytoene), C50-zeaxanthin, C50-caloxanthin, and C50-nostoxanthin (US20140170700); sarcinaxanthin, sarprenoxanthin, acyclic C50 carotenoid bacterioruberin, C50-canthaxanthin, C50-lycopene, C50-phytoene (Li et al., Scientific Reports volume 9, Article number: 2982 (2019)). See also Pfander, Pure and Applied Chemistry, 66(10-11):2369-2374 (1994).

Those skilled in the art are aware of a variety of technologies for producing carotenoid compounds. See, for example, Mezzomo et al., (2016) Carotenoids Functionality, Sources, and Processing by Supercritical Technology: A Review in J. Chemistry Volume 2016:1. In some embodiments, carotenoid compounds may be isolated from an organism (e.g., a plant or microbe) that has produced it. In some such embodiments, such plant or microbe may have been developed and/or cultivated by man. In some embodiments, a plant or microbe may be a natural plant or microbe. In some embodiments, a plant or microbe may be an engineered plant or microbe (e.g., a plant or microbe engineered to be a carotenoid-compound-synthesizing plant or microbe as described herein).

Those skilled in the art are aware of various plant and/or microbe sources that have been or can be engineered to be carotenoid-compound-synthesizing plants or microbes as described herein. See, for example, WO2016/102342.

In some embodiments, a carotenoid compound may be isolated from a plant or microbial source (e.g., from a cultivar or culture thereof). Those skilled in the art are aware of a variety of technologies for processing plant and/or microbial cells or tissues, for example, to prepare extracts thereof and/or to isolate components thereof and/or compounds therefrom.

Alternatively, or additionally, in some embodiments, a carotenoid compound may be partly or wholly prepared in vitro (e.g., by chemical and/or enzymatic synthesis, or a combination thereof), and may optionally be further isolated and/or purified as is known in the art.

A variety of technologies are known in the art that can be used to prepare extracts of cells or organisms that produce relevant carotenoid compounds, and/or to isolate extracts, components, or compounds therefrom, or to process (e.g., to isolate and/or purify one or more carotenoid compounds from) in vitro carotenoid synthesis systems. To give but a few examples, such technologies may include, for example, one or more of organic extraction, vacuum concentration, chromatography, and so on.

Those skilled in the art are aware that various lower order (shorter carbon chain) carotenoids such as α-carotene, β-carotene, γ-carotene, δ-carotene, ε-carotene, lutein, zeaxanthin, canthaxanthin, fucoxanthin, astaxanthin, antheraxanthin, and violaxanthin are synthesized by modification of the ends of lycopene through cyclization or oxidation. Higher order (more carbon atoms) carotenoids can be prepared, e.g., using in vitro methods or in vivo or ex vivo methods (e.g., via natural or genetically modified organisms). For example, C50 carotenoids can be synthesized in vitro by the addition of two dimethylallyl pyrophosphate (DMAPP) molecules to C(2) and C(2′) of the respective C40 carotenoid, or extracted from an organism (e.g., wild-type or engineered) that synthesizes the C50 carotenoid (e.g., a C50-carotenoid-compound-synthesizing microbe). See, e.g., Milon et al., Helv. Chim. Acta 69, 12-24 (1986); Karrer et al., Helv. Chim. Acta 34, 28-33 (1951); Furubayashi et al., Nat Commun. 2015; 6: 7534; Tobias and Arnold, Biochim Biophys Acta. 2006 February; 1761(2):235-46; Henke et al., (Jun. 14th 2017). Carotenoid Production by Corynebacterium: The Workhorse of Industrial Amino Acid Production as Host for Production of a Broad Spectrum of C40 and C50 Carotenoids, Carotenoids, Dragan J. Cvetkovic and Goran S. Nikolic, Intech Open, DOI: 10.5772/67631. Available from: intechopen.com/books/carotenoids/carotenoid-production-by-corynebacterium-the-workhorse-of-industrial-amino-acid-production-as-host-f; Henke et al., (2017). C50 Carotenoids: Occurrence, Biosynthesis, Glycosylation, and Metabolic Engineering for their Overproduction. In, Bio-pigmentation and Biotechnological Implementations, pp. 107-126. doi:10.1002/9781119166191.ch5; Fernandes, Introductory Chapter: Carotenoids—A Brief Overview on Its Structure, Biosynthesis, Synthesis, and Applications. In Progress in Carotenoid Research. (2018) doi: 10.5772/intechopen.79542; Heider et al., Appl Microbiol Biot 2014; 98(10):4355e68; Wang et al., Biotechnol Adv 2007; 25(3):211e22; Niu et al., Synthetic and Systems Biotechnology 2 (2017) 167e175; Li et al., Scientific Reports volume 9, Article number: 2982 (2019); US20050260699; US20140170700; US20040091958; US20090197321; and others.

In some embodiments, one or more carotenoid compounds for use in accordance with the present disclosure can be provided as purified, or substantially purified, molecules, or as less purified (e.g., enriched) extracts of an organism that produces a carotenoid.

In some embodiments, a preparation that is or comprises one or more carotenoid compounds is incorporated into or otherwise used to generate a pharmaceutical composition as described herein, that, when administered to a subject, delivers a carotenoid compound thereto.

In some embodiments, a carotenoid preparation or carotenoid composition (e.g., a pharmaceutical composition that comprises or delivers a carotenoid compound) comprises at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, or 99% w/w carotenoid compounds.

In some embodiments, compositions for use in accordance with the present disclosure are pharmaceutical compositions, e.g., for oral administration. Pharmaceutical compositions typically include an active agent (e.g., a carotenoid compound, such as a C50 carotenoid compound, or a source thereof), and a pharmaceutically acceptable carrier. Certain exemplary pharmaceutically acceptable carriers include, for instance saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

In some embodiments, a pharmaceutical composition for use in accordance with the present disclosure may include and/or may be administered in conjunction with, one or more supplementary active compounds; in certain embodiments, such supplementary active agents can include ginger, curcumin, probiotics (e,g, probiotic strains of one or more of the following genera: Lactobacillus, Bifidobacterium, Saccharomyces, Enterococcus, Streptococcus, Pediococcus, Leuconostoc, Bacillus, and/or Escherichia coli (see Fijan, Int J Environ Res Public Health. 2014 May; 11(5): 4745-4767); prebiotics (nondigestible food ingredients that help support growth of probiotic bacteria, e.g., fructans such as fructooligosaccharides (FOS) and inulins, galactans such as galactooligosaccharides (GOS), dietary fibers such as resistant starch, pectin, beta-glucans, and xylooligosaccharides (Hutkins et al., Curr Opin Biotechnol. 2016 February; 37: 1-7)) and combinations thereof.

Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include oral administration.

Methods of formulating suitable pharmaceutical compositions are known in the art, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). Oral compositions generally include an inert diluent or an edible carrier. To give but a few examples, in some embodiments, an oral formulation may be or comprise a syrup, a liquid, a tablet, a troche, a gummy, a capsule, e.g., gelatin capsules, a powder, a gel, a film, etc.

In some embodiments, pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of a pharmaceutical composition. In some particular embodiments, a pharmaceutical composition can contain, e.g., any one or more of the following inactive ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. In some embodiments, the compositions can be taken as-is or sprinkled onto or mixed into a food or liquid (such as water).

In some embodiments, a carotenoid composition that may be administered to subjects as described herein may be or comprise an ingestible item (e.g., a food or drink) that comprises (e.g., is supplemented with one or more carotenoids. In some embodiments, a useful composition may contain one or more carotenoid compounds at a level higher than that at which the carotenoid compound is found in components thereof, and/or higher than normally present in the relevant foods or beverages, e.g., to provide a level of carotenoids that is sufficient to provide a therapeutic effect as described herein.

In some embodiments, a food can be or comprise one or more of bars, candies, baked goods, cereals, salty snacks, pastas, chocolates, and other solid foods, as well as liquid or semi-solid foods including yogurt, soups and stews, and beverages such as smoothies, shakes, juices, and other carbonated or non-carbonated beverages. In some embodiments, foods are provided with carotenoids already included therein; in some embodiments, foods are prepared by a subject by mixing in carotenoids.

Compositions can be included in a kit, container, pack, or dispenser, together with instructions for administration or for use in a method described herein.

Those skilled in the art, reading the present disclosure, will appreciate that, in some embodiments, a carotenoid composition as described herein may be or comprise one or more cells, tissues, or organisms (e.g., plant or microbe cells, tissues, or organisms) that produce (e.g., have produced, and/or are producing) a relevant compound. In some embodiments, such cells, tissues, or organisms may have previously produced the relevant carotenoid; in some embodiments, such cells, tissues or organism are producing the carotenoid compound(s).

In some embodiments, carotenoid compositions may include cells, tissues, and/or organisms that have been killed (e.g., heat killed). Alternatively, in some embodiments, carotenoid compositions may include cells, tissues, and/or organisms that are viable or alive.

In some embodiments, methods of treatment as described herein involve administering one or more viable or living carotenoid-compound-synthesizing cells, tissues, or organisms. In some such embodiments, the cells, tissues, or organisms are microbial cells and are administered according to a regimen that achieves population of the subject's microbiome with administered cells.

In some embodiments, a carotenoid composition as described herein comprises and/or is formulated through use of one or more cell cultures and/or supernatants or pellets thereof, and/or a powder formed therefrom.

Those skilled in the art will appreciate that, in some embodiments, technologies for preparing carotenoid compositions and/or preparations, and/or for preparing one or more carotenoid compositions (and particularly for preparing pharmaceutical compositions) may include one or more steps of assessing or characterizing a compound, preparation, or composition, e.g., as part of quality control. In some embodiments, if an assayed material does not meet pre-determined specifications for the relevant assessment, it is discarded. In some embodiments, if such assayed material does meet the pre-determined specifications, then it continues to be processed as described herein.

Methods of Identifying and/or Characterizing

Among other things, the present disclosure provides systems that permit assessment of one or more agent characteristics relevant for usefulness as described herein. In some embodiments, technologies for identifying and/or characterizing agents as described herein may involve comparisons with an appropriate reference (e.g., with a positive control references and/or with a negative control reference). In some embodiments, a reference may be or comprise a historical reference; in some embodiments, a reference may be or comprise a contemporaneous reference.

In some embodiments, provided technologies may be useful for screening test agents, e.g., that may be or comprise one or more polypeptides, peptides, inorganic or organic large or small molecules, or compositions that include or deliver them, in order to identify agents useful in methods as described herein. Alternatively, or additionally, in some embodiments, provided technologies may be useful to characterize one or more agents, for example, during development and/or commercialization of such agent, or a pharmaceutically acceptable composition thereof.

As used herein, “small molecule” refers to small organic or inorganic molecules of molecular weight below about 3,000 Daltons. In general, small molecules may have a molecular weight of less than 3,000 Daltons (Da). The small molecules can be, e.g., from at least about 100 Da to about 3,000 Da (e.g., between about 100 to about 3,000 Da, about 100 to about 2500 Da, about 100 to about 2,000 Da, about 100 to about 1,750 Da, about 100 to about 1,500 Da, about 100 to about 1,250 Da, about 100 to about 1,000 Da, about 100 to about 750 Da, about 100 to about 500 Da, about 200 to about 1500, about 500 to about 1000, about 300 to about 1000 Da, or about 100 to about 250 Da).

Those skilled in the art will appreciate that in some embodiments, and particularly in screening embodiments, provided technologies may be utilized to identify (e.g., to screen) and/or characterize a plurality of agents. In some embodiments, such plurality is or comprises reasonably comparable agents (e.g., one or more particular small molecule compounds and a plurality of analogs thereof); in some embodiments, a plurality of agents is or comprises a plurality of natural products (e.g., carotenoid compounds such as C50 carotenoid compounds) and/or one or more analogs thereof. In some embodiments, a plurality of agents is or comprises a combinatorial library of small molecule compounds. Combinatorial techniques suitable for synthesizing small molecules are known in the art, e.g., as exemplified by Obrecht and Villalgordo, Solid-Supported Combinatorial and Parallel Synthesis of Small-Molecular-Weight Compound Libraries, Pergamon-Elsevier Science Limited (1998), and include those such as the “split and pool” or “parallel” synthesis techniques, solid-phase and solution-phase techniques, and encoding techniques (see, for example, Czarnik, Curr. Opin. Chem. Bio. 1:60-6 (1997)). In addition, a number of small molecule libraries are commercially available. A number of suitable small molecule test compounds are listed in U.S. Pat. No. 6,503,713, incorporated herein by reference in its entirety.

In some embodiments, provided technologies may be used to screen and/or assess a plurality of agents that cover a variety of functions such as charge, aromaticity, hydrogen bonding, flexibility, size, length of side chain, hydrophobicity, and rigidity.

In some embodiments, libraries screened as described herein can comprise a variety of types of test compounds. In some embodiments, a given library can comprise a set of structurally related or unrelated test compounds. In some particular embodiments, a library may comprise a set of peptides or peptidomimetic molecules. In some embodiments, a library may comprise carotenoid compounds, e.g., a naturally-occurring or synthetic carotenoid, e.g., a C35, C40, C45, C50, C55, or C60 carotenoid.

In some embodiments, provided technologies are utilized to assess a set of agents related to one another by systematically alteration of the structure of a first agent; in some such examples, the first agent may be or comprise a compound of known activity (e.g., a carotenoid compound such as a C50 carotenoid compound).

In some embodiments, technologies provided herein utilize or generate correlations between structural features and presence or absence (or level) of biological activity of interest—i.e., structure-function relationships. In some instances, structure-function relationships may be defined empirically; in some embodiments, structure-function relationships may be defined through utilization of computer modeling and/or analysis prediction methodologies.

In some embodiments, a food aversion assay as described herein is used. In some embodiments, a test sample is, or is derived from (e.g., a sample taken from) an in vivo model of a disorder as described herein. For example, a test compound is applied to a test sample comprising one or more C. elegans organisms, and one or more effects of the test compound is evaluated. For example, the ability of a test compound to counteract food aversion in the presence of a chemotherapeutic agent or other nausea-inducing stimulus can be tested. Alternatively, effects on a reporter gene, e.g., ABC transporter gene fusion pgp-5p::gfp, can be evaluated by detecting alterations in GFP fluorescence. As one of skill in the art will appreciate, other reporters could readily be used.

In some embodiments, a compound can be screened by a method described herein to determine whether it can reduce nausea (e.g., have anti-nausea activity), vomiting (e.g., have anti-emesis activity), and/or food aversion in a system (e.g., an animal model, e.g., C. elegans). In some embodiments, a compound that is determined to reduce nausea (e.g., have anti-nausea activity), vomiting (e.g., have anti-emesis activity), and/or food aversion in a system (e.g., an animal model, e.g., C. elegans) can be considered a candidate compound. A candidate compound that has been screened e.g., in a system having features associated with nausea, vomiting, and/or food aversion, e.g., an in vivo model of a disease, disorder, or condition associated with nausea and/or vomiting, e.g., C. elegans, and determined to have a desirable effect on nausea, vomiting, and/or food aversion can be considered a candidate therapeutic agent. In some embodiments, a candidate therapeutic agent can be tested in a larger animal model or in a clinical setting. Candidate therapeutic agents, once screened in a clinical setting, can be therapeutic agents. Candidate compounds, candidate therapeutic agents, and therapeutic agents can be optionally optimized and/or derivatized, and formulated with physiologically acceptable excipients to form pharmaceutical compositions.

A compound can be assessed by a method described herein to determine whether it can reduce nausea (e.g., have anti-nausea activity), vomiting (e.g., have anti-emesis activity), and/or food aversion in a system (e.g., an animal model, e.g., C. elegans). In some embodiments, the present disclosure provides a method for assessing a compound (e.g., a carotenoid) to determine its ability to reduce nausea (e.g., anti-nausea activity), vomiting (e.g., anti-emesis activity), and/or food aversion in a system (e.g., an animal model, e.g., C. elegans). In some embodiments, a method of assessing a compound to determine its ability to reduce nausea (e.g., anti-nausea activity), vomiting (e.g., anti-emesis activity), and/or food aversion in a system (e.g., an animal model, e.g., C. elegans) is part of an assay (e.g., a release test, a stability test, an efficacy test, etc.) performed for approval or maintenance of approval from a regulatory body (e.g., United States Food and Drug Administration, European Medicines Agency, etc.). In some embodiments, a method of assessing a compound (e.g., a carotenoid) to determine its ability to reduce nausea (e.g., anti-nausea activity), vomiting (e.g., anti-emesis activity), and/or food aversion in a system (e.g., an animal model, e.g., C. elegans) is part of a method of manufacture. In some embodiments, an assessment can be performed as part of a method of screening. In some embodiments, a system can be an animal system. In some embodiments, an animal system is a model system, e.g., C. elegans, cats, dogs, apes, or pigs.

Assessed compounds determined to reduce nausea (e.g., have anti-nausea activity), vomiting (e.g., have anti-emesis activity), and/or food aversion in a system (e.g., an animal model, e.g., C. elegans) can be systematically altered, e.g., using rational design, to optimize binding affinity, avidity, specificity, or another parameter. Such optimization can also be screened and/or assessed using the methods described herein. In some embodiments, a method includes screening a first library of compounds using one or more steps known in the art and/or described herein, e.g., identifying one or more hits in a library, subjecting hits to systematic structural alteration to create a second library of compounds structurally related to a hit, screening a second library using the methods described herein, or a combination thereof. Thus, in one embodiment, a method includes screening a first library of compounds using one or more steps known in the art and/or described herein, e.g., identifying one or more hits in a library, subjecting hits to systematic structural alteration to create a second library of compounds structurally related to a hit, screening a second library using the methods described herein, or a combination thereof.

An assessed compound determined to reduce nausea (e.g., have anti-nausea activity), vomiting (e.g., have anti-emesis activity), and/or food aversion in a system (e.g., an animal model, e.g., C. elegans) can be considered a candidate therapeutic compounds useful in reducing nausea, vomiting, and/or food aversion as described herein. A variety of techniques useful for determining the structures of compounds can be used in methods described herein, e.g., NMR, mass spectrometry, gas chromatography equipped with electron capture detectors, fluorescence and absorption spectroscopy. The present disclosure provides the insight that compounds determined to reduce nausea (e.g., have anti-nausea activity), vomiting (e.g., have anti-emesis activity), and/or food aversion in a system (e.g., by the methods described herein) can be used in method of treatment, prevention, or delay of development or progression of a disease, disorder and/or condition described herein. The present disclosure provides the insight that compounds determined to reduce nausea (e.g., have anti-nausea activity), vomiting (e.g., have anti-emesis activity), and/or food aversion in a system (e.g., by the methods described herein) can be used in method of treating nausea, vomiting, and/or a food version described herein.

An assessed compound determined to reduce nausea (e.g., have anti-nausea activity), vomiting (e.g., have anti-emesis activity), and/or food aversion in a system (e.g., C. elegans) can be assessed in a second system (e.g., a large animal, e.g., ape, monkey, cat, dog, pig, etc.). An animal can be monitored for a change in due to presence of a compound, e.g., for reduced nausea or food aversion. In some embodiments, a system is a human, e.g., a human with CINV or RINV, and a parameter is reduced severity, frequency, or duration of nausea, vomiting, and/or food aversion.

EXAMPLES

Embodiments provided herein are exemplified in the following examples, which do not limit the scope of the disclosure or claims.

Materials and Methods

The following materials and methods were used in the Examples below.

Strains

N2 Bristol was the wildtype strain used.

The following strains and mutant alleles were used:

SJ4100 [zcIs13[hsp-6::GFP],

WE5172 [ajIs1(pgp-5::gfp)X],

JG16 [eft-3(q145)/hT2[bli-4(e937) let-? (q782) qIs48] (I; III); ajIs1(pgp-5::gfp)X],

JG20 [ajs1 (pgp-5::gfp); agEX(pvha-6::mcherry::zip-2+myo-2::gfp)],

AY101 [acls101[F35E12.5P::GFP+rol-6(su1006)]

SJ4005 [zcIs4 [hsp-4::GFP] V],

SJ4100 (zcIs13[hsp-6::GFP])

Growth and Handling of Microbes Used:

16S ribosomal sequence was amplified using specific primers and sequenced to identify the microbes. LB media as well as plates was used for culturing Kocuria rhizophila, Arthrobacter arilaitensis and Corynebacterium glutamicum and its mutants. 500 ml of overnight culture was seeded onto SK media plates and incubated at room temperature for 2 days before initiating the experiments. For experiments involving Kocuria rhizophila wildtype and mutants, Arthrobacter arilaitensis, and Corynebacterium glutamicum wildtype and as well as mutants, synchronized L1-larval stage animals grown until L4-larval stage or day one of adulthood in E. coli OP50 seeded plates and was washed in M9 buffer at least five times before transferring to the appropriate bacterial food.

Drug Treatments:

Hygromycin diluted in M9 solution to the desired concentration was added onto E. coli OP50 bacteria containing NGM plates. Stock solution of emetine or cisplatin was diluted in M9 and the desired concentration was added onto E. coli OP50 bacteria containing NGM plates. 750 μg/ml of K. rhizophila extracts was added onto E. coli OP50 bacteria containing NGM plates containing appropriate concentrations of hygromycin or cisplatin or emetine. For the xenobiotic experiments, synchronized L1-stage animals were dropped onto the drug containing plates and scored 4 days later.

RNAi Assays:

For RNAi assays synchronized L1 larval stage animals of the appropriate genotype were fed with appropriate RNAi clones until they reach day one of adulthood. Subsequently, the RNAi-treated animals were washed in M9 at least five times to remove the E. coli bacteria and transferred to K. rhizophila seeded plates or E. coli OP50 seeded plates.

Microscopy

Nematodes were mounted onto agar pads and images were acquired using a Zeiss AXIO Imager Z1 microscope fitted with a Zeiss AxioCam HRm camera and Axiovision 4.6 (Zeiss) software. All the fluorescence images shown within the same figure panel were collected together using the same exposure time. Images were converted to 8-bit image, thresholded and quantified using ImageJ. Student's t test was used determine statistical significance. Low-magnification bright-field and GFP fluorescence images were acquired using a Zeiss AxioZoom V16, equipped with a Hammamatsu Orca flash 4.0 digital camera, and using Axiovision ZEN software.

Multiple Alignment of Protein Sequences

Multiple alignments were performed using Clustal Omega software.

K. rhizophila EMS Mutagenesis Screen

Mutagenesis was performed by treatment of overnight culture of K. rhizophila in PBS solution with 50 mM EMS for 45 minutes at 37° C. Serial dilutions of the mutagenized K. rhizophila cultures were plated onto LB media plates and ˜2000 mutagenized bacterial colonies were picked and grown in LB solution. 500 ml of overnight culture was seeded onto SK media plates and incubated at room temperature for two days before initiating the experiments. Synchronized L1-larval stage in eft-3(q145); pgp-5p::gfp animals grown until L4-larval stage or day one of adulthood in E. coli OP50 seeded plates and was washed in M9 buffer at least five times before transferring to the K. rhizophila mutant bacterial food. The plates were visually screened after 24 hours for GFP induction.

Identification of EMS Induced Mutations by Whole Genome Sequencing

Genomic DNA extraction, library prep, Illumina MiniSeq sequencing and bioinformatics were all performed by The Sequencing Center located at Fort Collins, Colo.

Isolation of K. rhizophila Carotenoids

Carotenoid isolation from K. rhizophila was isolated as described (Giuffrida et al., 2016) with the following modifications. K. rhizophila cultures grown in LB solution was washed with equal volume of water after centrifugation at 4000 RPM for 15 min. After centrifugation to remove water, equal volume of acetone was added and centrifuged again at 4000 RPM for 15 min. After removal of acetone, the bacterial pellets were extracted with methanol at 65° C. in water bath after wrapping the samples with aluminum foil to protect from light. The samples were extracted with methanol multiple times until all the cells were bleached. The supernatant was filtered with Whatman filter paper No 1. Two-volumes of 15% sodium chloride was added to the methanol extract and after mixing equal volume of hexane was added. The yellow carotenoids were separated from the methanol-salt mix and accumulated in the hexane fraction. The hexane fraction was removed and washed at least three times with water. The hexane fraction was evaporated and the resultant carotenoid pellet was dissolved in methanol.

High Performance Liquid Chromatography

Crude methanolic extracts were separated over an Agilent Eclipse Plus C18 4.6×250 mm column with a 5-micron particle size using an Agilent 1200 HPLC equipped with a diode array detector, autosampler, column oven, solvent degasser, and binary pump. The mobile phases were (A) water vs. (B) methanol at a flow rate of 2 mL/min. The column was preequilibrated at 40° C. with 90% B prior to sample injection. Following injection, the column was washed isocratically for 5 min at 90% B before ramping to 100% B over 5 min. Eluate absorbance spectra were monitored from 300-700 nm.

Pull Down Experiments with Rat Liver

To identify carotenoid binding proteins, the protocol from Pilbrow et al., 2014 was used with the following modifications. Protein extracts were obtained by chopping ˜10 grams of adult rat liver into tiny pieces and homogenizing using Tissue-in T-PER tissue protein extraction reagent containing Roche protease inhibitors. Delipidation of the extract was done using methanol-chloroform. ˜10 mg of K. rhizophila carotenoids were incubated with 1 gram of protein extract for one hour at 22° C. Unbound carotenoids were removed by size-exclusion chromatography using Bio-spin P-6 (6K MWCO). The carotenoid bound protein extract was subjected to DEAE anion-exchange sepharose resin column pre-equilibrated with anion-exchange buffer A (0.05M dibasic sodium phosphate, pH 8.0) at 4° C. The sample was allowed to flow through the column by gravity and the column was washed with anion-exchange buffer A. Proteins were eluted with 0.5M NaCl in 0.5 ml volumes. The yellow fractions were pooled, dialyzed to remove salts and concentrated using Vivaspin centrifugal concentrator columns. The yellow fractions were separated on 4-12% Native PAGE and the yellow-orange band visible was excised and mass spectrometry was conducted for identifying proteins.

Putative “Decaprenoxanthin” Carotenoid Biosynthetic Cluster from Microbes

Putative carotenoid biosynthetic cluster of Leifsonia xyli (Lxx15630, Lxx15620, Lxx15610, Lxx15600, Lxx15590, and Lxx15580) (Monteiro-Vitorello et al., 2004), Microbacterium testaceum (MTES_3133, MTES_3132, MTES_3131, MTES_3130, MTES_3129, and MTES_3128) (Morohoshi et al., 2011), Cellvibrio gilvus (Celgi_1516, Celgi_1515, Celgi_1514, Celgi_1513, Celgi_1512, and Celgi_1511) (Christopherson et al., 2013), Cellulomonasfimi (Celf_3171, Celf_3170, Celf_3169, Celf_3168, Celf_3167, and Celf_3166) (Christopherson et al., 2013), Sanguibacter keddieii (Sked_12750, Sked_12760, Sked_12770, Sked_12780, Sked_12790, and Sked_12800) (Ivanova et al., 2009), Jonesia denitrificans (Jden_0342, Jden_0341, Jden_0340, Jden_0339, Jden_0338, and Jden_0337) (Pukall et al., 2009), Mycetocola manganoxydans (D9V29_RS08865, D9V29 RS08870, D9V29 RS08875, D9V29 RS08880, D9V29 RS08885, and D9V29_RS08890), Mycetocola miduiensis (BM197_RS02470, BM197_RS02475, BM197_RS02480, BM197_RS02485, BM197_RS02490, and BM197_RS02495), Cryobacterium psychrotolerans (BLQ39_RS02180, BLQ39_RS02185, BLQ39_RS02190, BLQ39_RS02195, BLQ39_RS02200, and BLQ39_RS02205), Subtercola boreus (B7R21_RS02695, B7R21_RS02700, B7R21_RS02705, B7R21_RS02710, B7R21_RS02715, and B7R21_RS02720), Herbiconiux solani (HSO01 S_RS07000, HSO01S_RS07005, HSO01S_RS07010, HSO01S_RS07015, HSO01S_RS07020, and HSO01S_RS07025), Microbacterium phyllosphaerae (D3H67_RS09120, D3H67_RS09125, D3H67_RS09130, D3H67_RS09135, D3H67_RS09140, and D3H67_RS09145), Leifsonia aquatica (N136_RS22055, N136_RS22060, N136_RS22065, N136_RS22070, N136_RS22075, and N136_RS22080), Microbacterium esteraromaticum (B4U78_RS09520, B4U78_RS09525, B4U78_RS09530, B4U78_RS09535, B4U78_RS09540, and B4U78_RS09545),

Plantibacter sp. H53 (A4X17_RS18565, A4X17_RS18570, A4X17_RS18575, A4X17_RS18580, A4X17_RS18585, and A4X17_RS18590), Curtobacterium sp. (ASF23_RS14315, ASF23_RS14320, ASF23_RS14325, ASF23_RS14330, ASF23_RS14335, and ASF23_RS14340), Microterricola pindariensis (GY24_RS04745, GY24_RS04750, GY24_RS04755, GY24_RS04760, GY24_RS04765, and GY24_RS04770), Frondihabitans sp. (EDF46_RS08000, EDF46_RS08005, EDF46_RS08010, EDF46_RS08015, EDF46_RS08020, and EDF46_RS08025), Salinibacterium xinjiangense (SAMN06296378_0676, SAMN06296378_0677, SAMN06296378_0678, SAMN06296378_0679, SAMN06296378_0680, and SAMN06296378_0681), Agromyces sp. (AVP42_RS01110, AVP42_RS01115, AVP42_RS01120, AVP42_RS01125, AVP42_RS01130, and AVP42_RS01135), Microbacterium barkeri (MBR4_RS00310, MBR4_RS00315, MBR4_RS00320, MBR4_RS00325, MBR4_RS00330, and MBR4_RS00335), Arthrobacter koreensis (BN2404_RS04370, BN2404_RS04375, BN2404_RS04380, BN2404_RS04385, BN2404_RS04390, and BN2404_RS04395), Cryobacterium roopkundense (GY21_RS00565, GY21_RS00570, GY21_RS00575, GY21_RS00580, GY21_RS00585, and GY21_RS00590), Microbacterium oxydans (RN51_RS07325, RN51_RS07330, RN51_RS07335, RN51_RS07340, RN51_RS07345, and RN51_RS07350), Arthrobacter luteolus (AL3_RS02570, AL3_RS02575, AL3_RS02580, AL3_RS02585, AL3_RS02590, and AL3_RS02595), Cryobacterium aureum (CJ028_RS03575, CJ028_RS03580, CJ028_RS03585, CJ028_RS03590, CJ028_RS03595, and CJ028_RS03600), Curtobacterium ammoniigenes (CAM01S_RS13455, CAM01S_RS13460, CAM01S_RS13465, CAM01S_RS13470, CAM01S_RS13475, and CAM01S_RS13480), Oerskovia enterophila (OJAG_RS08920, OJAG_RS08925, OJAG_RS08930, OJAG_RS08935, OJAG_RS08940, and OJAG_RS08945),

Microbacterium paraoxydans (SAMN04489809_1122, SAMN04489809_1123, SAMN04489809_1124, SAMN04489809_1125, SAMN04489809_1126, and SAMN04489809_1127), Agromyces subbeticus (H521_RS21795, H521_RS21800, H521_RS0106365, H521_RS0106370, H521_RS21805, and H521_RS0106380), Arthrobacter crystallopoietes (D477_RS18370, D477_RS18375, D477_RS18380, D477_RS18385, and D477_RS18390, D477_RS18395), Georgenia satyanarayanai (DSZ44_RS04745, DSZ44_RS04750, DSZ44_RS04755, DSZ44_RS04760, DSZ44_RS04765, and DSZ44_RS04770), Microbacterium trichothecenolyticum (RS82_RS03115, RS82_RS03120, RS82_RS03125, RS82_RS03130, RS82_RS03135, and RS82_RS03140), Arthrobacter woluwensis (C6401_RS03950, C6401_RS03955, C6401_RS03960, C6401_RS03965, C6401_RS03970, and C6401_RS03975), Promicromonospora kroppenstedtii (PROKR_RS13815, PROKR_RS13820, PROKR_RS13825, PROKR_RS13830, PROKR_RS13835, and PROKR_RS13840), Cellulomonas cellasea (Q760 RS04595, Q760_RS04600, Q760_RS04605, Q760_RS18340, Q760_RS04615, and Q760_RS04620), Agromyces cerinus (BUR99_RS12060, BUR99_RS12065, BUR99_RS12070, BUR99_RS12075, BUR99_RS12080, and BUR99_RS12085), Agreia pratensis (B9Y86_RS06900, B9Y86_RS06905, B9Y86_RS06910, B9Y86_RS06915, B9Y86_RS06920 and B9Y86_RS06925), Microbacterium laevaniformans (OR221_3062, OR221_3063, OR221_3064, OR221_3065, OR221_3066, and OR221_3067), Arthrobacter stackebrandtii (CVV67_17780, CVV67_17785, CVV67_17790, CVV67_17795, CVV67_17800, and CVV67_17805), Paeniglutamicibacter gangotriensis (ADIAG_RS03760, ADIAG_RS03765, ADIAG_RS03770, ADIAG_RS03775, ADIAG_RS03780, and ADIAG_RS03785), Microbacterium trichothecenolyticum (RS82_RS03115, RS82_RS03120, RS82_RS03125, RS82_RS03130, RS82_RS03135, and RS82_RS03140), Arthrobacter livingstonensis (CVV68_RS19330, CVV68_RS19335, CVV68_RS19340, CVV68_RS19345, CVV68_RS19350, and CVV68_RS19355), Demequina lutea (AOP76_RS09030, AOP76_RS09035, AOP76_RS09040, AOP76_RS09045, AOP76_RS09050, and AOP76_RS09055), Zhihengliuella halotolerans (CUR88_RS12685, CUR88_RS12690, CUR88_RS12695, CUR88_RS12700, CUR88_RS12705, and CUR88_RS12710), Paeniglutamicibacter antarcticus (BN2261_RS08280, BN2261_RS08285, BN2261_RS08290, BN2261_RS08295, BN2261_RS08300, and BN2261_RS08305), Janibacter melonis (EEW87_RS00715, EEW87_RS00720, EEW87_RS00725, EEW87_RS00730, EEW87_RS00735, and EEW87_RS00740), Microbacterium arborescens (DOU46_RS02280, DOU46_RS02285, DOU46_RS02290, DOU46_RS02295, DOU46_RS02300, and DOU46_RS02305), Agreia pratensis (B9Y86_RS06900, B9Y86_RS06905, B9Y86_RS06910, B9Y86_RS06915, B9Y86_RS06920, and B9Y86_RS06925), Agreia bicolorata (TZ00_RS04480, TZ00_RS04485, TZ00_RS19215, TZ00_RS04495, TZ00_RS04500, and TZ00_RS04505), Arthrobacter psychrochitiniphilus (CVS30_RS02785, CVS30_RS02790, CVS30_RS02795, CVS30_RS02800, CVS30_RS02805, and CVS30_RS02810), Microterricola pindariensis (GY24_RS04745, GY24_RS04750, GY24_RS04755, GY24_RS04760, GY24_RS04765, and GY24_RS04770), Microbacterium indicum (H576_RS15860, H576_RS0112930, H576_RS0112935, H576_RS0112940, H576_RS0112945, and H576_RS15865), Homoserinimonas sp. (DL891_RS01870, DL891_RS01875, DL891_RS01880, DL891_RS01885, DL891_RS01890, and DL891_RS01895), Cryobacterium levicorallinum (SAMN05216274_11068, SAMN05216274_11069, SAMN05216274_11070, SAMN05216274_11071, SAMN05216274_11072, and SAMN05216274_11073), Frigoribacterium sp. (EDF18_RS14355, EDF18_RS14360, crtI, EDF18_RS14370, EDF18_RS14375, and EDF18_RS14380), Cryobacterium luteum (SAMN05216281_10883, SAMN05216281_10884, SAMN05216281_10885, SAMN05216281_10886, SAMN05216281_10887, and SAMN05216281_10888), Cellulomonas carbonis (N868 RS13600, N868_RS13605, N868_RS13610, N868_RS13615, N868_RS13620, and N868_RS13625), Okibacterium fritillariae (B5X75_RS14075, B5X75_RS14080, B5X75_RS14085, B5X75_RS14090, B5X75_RS14095, and B5X75_RS14100), Glycomyces sambucus (BLS99_RS13650, BLS99_RS13655, BLS99_RS13660, BLS99_RS13665, BLS99_RS13670, and BLS99_RS13675), Krasilnikoviella flava (B5Y66_RS20515, B5Y66_RS20520, B5Y66_RS20525, B5Y66_RS20530, B5Y66_RS20535, and B5Y66_RS20540), Actinotalea ferrariae (N866_01505, N866_01510, N866_01515, N866_01520, N866_01525, and ubiA), Lysinimicrobium soli (AOM04_RS11780, AOM04_RS11785, AOM04_RS11790, AOM04_RS11795, AOM04_RS11800, and AOM04_RS11805), Luteimicrobium subarcticum (CLV34_RS08275, CLV34_RS08280, CLV34_RS08285, CLV34_RS08290, CLV34_RS08295, and CLV34_RS08300), Promicromonospora kroppenstedtii (PROKR_RS13815, PROKR_RS13820, PROKR_RS13825, PROKR_RS13830, PROKR_RS13835, and PROKR_RS13840), Tersicoccus phoenicis (BKD30_RS05860, BKD30_RS05865, BKD30_RS05870, BKD30_RS05875, BKD30_RS05880, and BKD30_RS05885), Sinomonas humi (LK10_RS09295, LK10_RS09300, LK10_RS09305, LK10_RS09310, and LK10_RS09315), Pseudarthrobacter phenanthrenivorans (RM50_RS01675, RM50_RS01680, RM50_RS01685, RM50_RS01690, RM50_RS01695, and RM50_RS01700), Acaricomes phytoseiuli (C501_RS0107225, C501_RS0107230, C501_RS0107235, C501_RS0107240, C501_RS0107245, and C501_RS0107250), Leucobacter musarum (AMS67_RS10795, AMS67_RS10800, AMS67_RS10805, AMS67_RS10810, AMS67_RS10815, and AMS67_RS10820), Ornithinimicrobium pekingense (K330_RS19765, K330_RS0107130, K330_RS19770, K330_RS19775, K330_RS0107145, and K330_RS0107150), Citricoccus sp. (CITRI_RS16000, CITRI_RS0102550, CITRI_RS0102555, CITRI_RS16005, CITRI_RS0102565, and CITRI_RS0102570), and Arthrobacter arilatensis (AARI_13710, AARI_13720, AARI_13730, AARI_13740, AARI_13760, and AARI_13750) (Monnet et al., 2010) are very similar have a similar size and show the same organization as in the genome of K. rhizophila. In Corynebacterium glutamicum (cg0723, cg0721, cg0720, cg0719, cg0718, and cg0717) (Kalinowski et al., 2003) and Corynebacterium efficiens (HMPREF0290_1086, HMPREF0290_1088, HMPREF0290_1089, HMPREF0290_1090, HMPREF0290_1091, and HMPREF0290_1092) (Nishio et al., 2003) the decaprenoxanthin producing gene cluster is similar in size and organization to that of K. rhizophila except for the insertion of an unrelated gene cg0722 between crtE and crtB in C. glutamicum or HMPREF0290_1088 in C. efficiens. Also, in Corynebacterium glutamicum, an additional carotenoid cluster (NCg10600, NCg10598, NCg10597, NCg10596, NCg10595, and NCg10594) in also present in the genome. In Kytococcus sedentarius, the genes responsible for carotenoid production (Ksed_13840, Ksed_13830, Ksed_13820, Ksed_13810, Ksed_13800) are arranged in the same cluster while Ksed_16070, which encodes for geranylgeranyl pyrophosphate synthase is located elsewhere in the genome (Sims et al., 2009).

In Brevibacterium mcbrellneri, the genes responsible for carotenoid production (HMPREF0183_0793, HMPREF0183_0794, HMPREF0183_0795, HMPREF0183_0796, and HMPREF0183_0797) are located in the same cluster while HMPREF0183_0437, which encodes for polyprenyl synthetase is located elsewhere in the genome.

In Beutenbergia cavernae, the genes responsible for carotenoid production (Bcav_3492, Bcav_3491, Bcav_3490, Bcav_3489, Bcav_3488) are located in the same cluster while Bcav_0970, which encodes for polyprenyl synthetase is located elsewhere in the genome (Land et al., 2009).

In Brachybacterium faecium (Bfae_04470, Bfae_04440, Bfae_04430, Bfae_04420, Bfae_04410, and Bfae_04400) (Lapidus et al., 2009) the carotenoid biosynthetic cluster is similar in size and organization to that of K. rhizophila except for the insertion of two unrelated genes Bfae_04460 and Bfae_04450 between Bfae_04470 and Bfae_04440.

In Cellulomonas flavigna (Cfla_2888, Cfla_2889, Cfla_2890, Cfla_2891, and Cfla_2892) (Abt et al, 2010) all the genes responsible for carotenoid production are present except for Cfla_2893 which is a likely pseudogene because of frame-shift mutation.

Decaprenoxanthin was the first C50 carotenoid discovered from Flavobacterium dehydrogenans (now known as Agromyces mediolanus (Liaaen-Jensen et al., 1968)).

Many bacteria including Agromyces mediolanus (Liaaen-Jensen et al., 1968), Aureobacterium sp. (Fukuoka et al., 2004), Arthrobacter glacialis (Arpin et al., 1975), Arthrobacter arilatensis (Sutthiwong et al., 2014), Cellulomonas biazotea (Weeks et al., 1980), Citriococcus sp, and Corynebacterium glutamicum (Krubasik et al., 2001) are known to produce decaprenoxanthin.

Example 1. Bacterial Carotenoids Suppress C. elegans Surveillance of Translational Deficits

Expression of particular suites of cytochrome p450, ABC transporter, UDP-glycosyl transferase genes among the approximately 500 C. elegans xenobiotic detoxification genes, for example, the C. elegans ABC transporter gene pgp-5, can be induced by toxin, RNAi, or mutational inhibition of ribosomal proteins, tRNA synthetases, and other genes implicated in translation (Govindan et al., 2015). Even when the defects in translation are limited to the germline, for example, in the C. elegans eft-3(q145) mutant, a mutation in the germline isoform of the translation elongation factor-1 (where translation in somatic cells and somatic development is normal, but translation in the germline cells is disabled and the germline does not proliferate), the expression of the ABC transporter gene fusion pgp-5p::gfp was strongly induced in the intestine when the animals are fed the benign E. coli OP50 (FIG. 5A). Feeding K. rhizophila rather than E. coli to C. elegans eft-3(q145); pgp-5p::gfp disrupted the normal induction of pgp-5p::gfp (FIGS. 5A&B). K. rhizophila is a gram-positive coccus of the phylum Actinobacteria, a clade rich in drug biosynthetic pathways. K. rhizophila species are found in various ecological niches including soils and the C. elegans gut microbiome in natural populations of nematodes from orchards (Felix et al., 2010). K. rhizophila species are normal inhabitants of skin and mucous membrane of human and animals, but can be associated with human infections.

To establish that K. rhizophila can suppress the surveillance of a range of defects in translation, the induction of xenobiotic detoxification response to RNAi of other ribosomal proteins was tested. Synchronized L1-larval stage pgp-5p::gfp animals were fed on E. coli expressing either rpl-1 dsRNA or vrs-2 dsRNA, which inhibit the production of the C. elegans RPL-1 ribosomal protein and the VRS-2 tRNA synthetase. When these animals reached adulthood, they were transferred to either E. coli OP50 containing plates or K. rhizophila seeded plates and the scored for GFP induction after 24 hours. In animals fed on either vrs-2 dsRNA or rpl-1 dsRNA and transferred to E. coli OP50 containing plates, pgp-5p::gfp was induced. By contrast, in animals fed on either rpl-1 dsRNA or vrs-2 dsRNA and transferred to K. rhizophila plates, pgp-5p::gfp expression was abrogated (FIGS. 5C&D). The suppression of surveillance pathways by K. rhizophila is specific for translation stress because K. rhizophila does not suppress the induction of mitochondrial stress response or endoplasmic reticulum stress response (Govindan et al., 2015).

To identify the K. rhizophila pathways responsible for the inhibition of pgp-5p::gfp induction in a C. elegans strain carrying a genetic deficit in translation (eft-3(q145), a forward genetic screen was conducted for K. rhizophila mutant strains that are defective in the inhibition of pgp-5p::gfp induction. About 2000 individual K. rhizophila strains that grew normally on bacterial LB plates after EMS mutagenesis were fed to eft-3(q145); pgp-5p::gfp animals. These 2000 individual wells of distinct K. rhizophila mutant strains were screened for mutant bacterial strains that failed to suppress pgp-5p::gfp induction in the eft-3(q145); pgp-5p::gfp animals. Six K. rhizophila mutant strains were identified that failed to induce pgp-5p::gfp in eft-3(q145); pgp-5p::gfp animals (FIGS. 5A&E). Visual inspection revealed that all these mutant strains had defects in colony pigmentation compared to wild type K. rhizophila (FIG. 5F). Wildtype K. rhizophila is yellow, whereas the six mutant colonies were red or white or orange. Using this discoloration phenotype, we screened ˜500,000 bacterial colonies generated by EMS mutagenesis visually for mutants with a discoloration phenotype. We isolated 71 mutants that were discolored (FIG. 5G). The 71 mutants along with 25 control non-discolored mutants generated in the same EMS mutagenesis were tested on eft-3(q145); pgp-5p::gfp animals and scored for GFP induction. All 71 discoloration mutants failed to suppress pgp-5p::gfp induction in the C. elegans eft-3(q145) mutant, whereas the 25 normally colored strains suppressed pgp-5p::gfp induction (FIG. 6A; FIG. 1A-C).

Genome sequencing of 23 EMS-mutagenized K. rhizophila mutants that failed to suppress pgp-5p::gfp induction in the eft-3(q145) mutant revealed that each carried a mutation in one of six carotenoid biosynthetic cluster genes (FIG. 6B; FIG. 1C-E).

Carotenoids are yellow to red colored pigments, which are produced by a terpenoid biosynthetic pathway. The genome of K. rhizophila contains an operon that encodes predicted carotenoid (crt) biosynthetic genes (Takarada et al., 2008). These include crtE (KRH_20850; encoding GGPP synthase), crtB (KRH_20840; encoding phytoene synthase), crtI (KRH_20830; encoding phytoene desaturase), crtEb (KRH_20800; encoding lycopene elongase), crtYe (KRH_20820; encoding C50 carotenoid epsilon cyclase) and crtYf(KRH_20810; encoding C₅₀ carotenoid epsilon cyclase) (FIG. 1C-E). The reaction catalyzed by GGPP synthase CrtE, phytoene synthase CrtB and phytoene desaturase CrtI based on orthology are predicted to mediate steps in the production of lycopene (Klassen et al., 2010; Krubasik et al., 2001). CrtEb and CrtYe/f cyclases catalyze the biosynthesis of C₅₀ carotenoid from lycopene. C₅₀ carotenoids are rare in nature and very few of them have been characterized (Krubasik et al., 2001a; Krubasik et al., 2001b; Norgard et al., 1970; Tao et al., 2007; Netzer et al., 2010).

Even though there are several microbes that contain the CrtEb and CrtYe/f genes (FIG. 7), to date the only genetically and biochemically well-characterized C₅₀ carotenoid is decaprenoxanthin from C. glutamicum (Heider et al., 2012). In C. glutamicum, the enzymes CrtEb, CrtYe, and CrtYf convert lycopene to the C₅₀ carotenoid decaprenoxanthin (Krubasik et al., 2001a; Krubasik et al., 2001b) (FIG. 1E). This reaction in carried out in two steps: CrtEb catalyzes the elongation of C₄₀ acyclic lycopene to acyclic C₅₀ carotenoid flavuxanthin (Krubasik et al., 2001a; Krubasik et al., 2001b). The products of CrtYe and CrtYf, which combine to form a C₅₀ cyclase then catalyze the conversion of C₅₀ carotenoid flavuxanthin to decaprenoxanthin (Krubasik et al., 2001a; Krubasik et al., 2001b). K. rhizophila crtYe and crtYf are 38% and 34% identical to C. glutamicum crtYe and crtYf respectively. Therefore, the yellow pigment produced by K. rhizophila may belong to the decaprenoxanthin C₅₀-subfamily.

From the genetic screen of K. rhizophila, multiple mutations were obtained in crtI, which encodes phytoene desaturase, including six missense mutations (e21, e11, e23, e14, e5, and e13) and two nonsense mutations (e15 and e10) (FIG. 1C-E). CrtI catalyzes the conversion of the non-colored phytoene to lycopene, which is red. All these crtI mutants were white colored colonies (FIG. 6B; FIG. 1C) similar to C. glutamicum ΔcrtI mutant (Heider et al., 2012) (FIG. 6C) and thus likely lacked lycopene synthesis. The six missense mutations were in highly conserved residues suggesting that they may be important for protein function (FIG. 8). The e4, e6, and e8 were missense mutations in the crtB gene, which encodes phytoene synthase (FIGS. 1D&E). All these missense mutations were in highly conserved residues suggesting that they may be important for protein function (FIG. 9). These mutants produced white bacterial colonies (FIG. 6B; FIG. 1C) as was observed in C. glutamicum ΔcrtB mutant (Heider et al., 2012) (FIG. 6C). Four mutations were obtained in crtEb; two nonsense mutations (e16 and e17) and two missense mutations (e3 and e19) in highly conserved residues (FIG. 1D; FIG. 10). Mutations in crtEb were likely to be defective in the conversion of lycopene to flavuxanthin (FIG. 1E). These mutants formed pale red colonies (FIG. 6; FIG. 1B) probably because of accumulation of lycopene (but not flavuxanthin) as was seen in C. glutamicum ΔcrtEb mutant (Heider et al., 2012) (FIG. 6C). e17 was an early stop mutation in crtEb which is predicted to produce a truncated protein of just 13 amino acids (FIG. 10). Mutations in crtYe and crtYf were defective in the last step; the homologs of these genes catalyze synthesis of decaprenoxanthin. These mutants produced pale red to orange colonies (FIG. 6B). Interestingly, the C. glutamicum ΔcrtY mutant accumulated flavuxanthin and also exhibited a pale orange to red color (Heider et al., 2012) (FIG. 6C).

Because C. glutamicum ATCC13032 is known to produce decaprenoxanthin, whether feeding C. glutamicum ATCC13032 feeding would suppress pgp-5p::gfp induction was tested in eft-3(q145);pgp-5p::gfp animals. Feeding wild type C. glutamicum ATCC13032 to eft-3(q145);pgp-5p::gfp animals suppressed pgp-5p::gfp induction (FIGS. 2A&B). In C. glutamicum ATCC13032, the carotenoid gene cluster CrtE-cg0722-CrtBIYeYfEb mediates decaprenoxanthin biosynthesis (Heider et al., 2012). Whether C. glutamicum ATCC13032 deletion mutants in crtY, crtEb, crtI, and, crtB, which are known to lack decaprenoxanthin production (Heider et al., 2012), could suppress pgp-5p::gfp induction in eft-3(q145);pgp-5p::gfp animals was tested. Animals fed on ΔcrtY, ΔcrtEb, ΔcrtI, ΔcrtB C. glutamicum showed normal pgp-5p::gfp induction, unlike the same strain grown on wild type C. glutamicum (FIGS. 2A&B).

The pigmented bacterium Arthrobacter arilaitensis is known to produce decaprenoxanthin (Monnet et al., 2010); experiments tested whether feeding A. arilaitensis feeding would suppress pgp-5p::gfp induction in eft-3(q145);pgp-5p::gfp animals. Feeding A. arilaitensis to eft-3(q145);pgp-5p::gfp animals also suppressed pgp-5p::gfp expression (FIG. 2C). Thus, C. glutamicum, A. arilaitensis, or K. rhizophila produce a pigmented carotenoid that mediates suppression of translational surveillance to the induction of ABC transporter detoxification responses.

Carotenoids such as decaprenoxanthin are lipophilic molecules that localize to the cell membrane and can be easily extracted in non-polar solvents. K. rhizophila cultures were extracted with such solvents (FIG. 11). TLC analysis of the extract revealed the presence of yellow-orange pigment (FIG. 2D). HPLC analysis of a methanol extract from K. rhizophila was complexed with at least six different components (FIG. 11; FIG. 2E). These peaks were named according to the time of elution times as peak 1 (5.4 min), peak 2 (6.3), peak 3 (6.5), peak 4 (7.2), peak 5 (7.8), and peak 6 (8.7) (FIG. 2E). Absorbance spectra of the elution peaks revealed absorption maxima at 420, 440 and 470 nm which is similar to the published absorption spectra of decaprenoxanthin from Arthrobacter (Giuffrida et al., 2016; Sutthiwong et al., 2014).

To analyze the carotenoid production in different K. rhizophila mutants, carotenoids were extracted from crtI(e10), crtEb(e17), crtYe(e22), and crtYf(e18), which are nonsense mutant alleles and crtB(e6) which is a missense mutant. Spectrophotometric analysis of methanol extracts from K. rhizophila wildtype showed absorption maxima of 415-425 nm, whereas the crtEb(e17) extract showed absorption maxima at 445-455 nm (FIG. 12). K. rhizophila crtEb(e17) and crtYe(e22) mutant methanol extract show similar absorption spectra while the extract from K. rhizophila crtI(e10) and crtb(e6) showed no absorption at all. The methanol extracts from crtYf(e18) showed two separate absorption peaks one at ˜400 nm and another minor one at ˜500 nm.

The ability of crude methanol extracts from wildtype K. rhizophila, containing carotenoids, to suppress the GFP induction in eft-3(q145);pgp-5p::gfp animals was also tested. Animals fed on E. coli with added wildtype K. rhizophila extract exhibited significantly reduced pgp-5p::gfp expression compared to eft-3(q145);pgp-5p::gfp animals fed on E. coli with control methanol extract (FIG. 2F). The extract wild type K. rhizophila methanol extract could rescue the suppression of C. elegans surveillance defect of K. rhizophila carotenoid biosynthetic mutants: when eft-3(q145);pgp-5p::gfp animals were fed on K. rhizophila crtEb(e17), crtB(e6) or crtI(e10) mutants supplemented with K. rhizophila wildtype extract, GFP was not induced while in the animals fed on control extract, the GFP expression was induced by the C. elegans eft-3 mutation (FIG. 2G).

One trivial explanation for the failure of the K. rhizophila carotenoid mutants to suppress pgp-5p::gfp in a translation-defective C. elegans mutant would be that these K. rhizophila pigmentation mutants might induce pgp-5p::gfp even in a wildtype C. elegans background. To test this possibility, wild type C. elegans carrying the pgp-5p::gfp fusion gene were fed on K. rhizophila crtEb(e17), crtYe(e22), crtYf(e18), crtB(e6) or crtI(e10) mutants. The results showed that K. rhizophila wildtype and carotenoid mutants did not induce pgp-5p::gfp (FIG. 3A). Another possible interpretation was that K. rhizophila feeding might induce other stress responses in C. elegans that somehow “distract” the animal from surveillance of translation. The effects of induction of other GFP fusion reporters of stress was tested in wild type and various K. rhizophila mutants. hsp-4p::gfp and hsp-6p::gfp are reporters of endoplasmic reticulum unfolded protein response (UPR^(ER)) and mitochondrial unfolded protein response (UPR^(mito)) respectively (Yoneda et al., 2004; Calfon et al., 2002). clec-60 is a C-type lectin/CUB domain protein induced by the gram-positive pathogens, S. aureus and M. nematophilum (O'Rourke et al., 2006). F35E12.5p::GFP is a CUB domain protein induced by Y. pestis, M. nematophilum and P. aeruginosa (O'Rourke et al., 2006; Troemel et al., 2006; Bolz et al., 2010). C. elegans hsp-4p::gfp, hsp-6p::gfp, F35E12.5p::GFP, and clec-60p::gfp were fed on K. rhizophila wildtype and carotenoid mutants. The K. rhizophila did not induce hsp-4p::gfp expression (FIG. 13A). Similarly, K. rhizophila wildtype or carotenoid mutant did not induce hsp-6p::gfp expression (FIG. 13B). K. rhizophila wildtype or carotenoid mutants does not induce F35E12.5p::GFP (FIG. 13D). But K. rhizophila wildtype or carotenoid mutants induced clec-60::GFP (FIG. 13C), which is induced by gram positive bacteria. Because K. rhizophila is also a gram-positive bacterium, the induction of clec-60 is most likely an immune response to a pathogen.

To address whether the effect of K. rhizophila feeding on the suppression of pgp-5p::gfp induction is reversible, eft-3(q145);pgp-5p::gfp animals fed on K. rhizophila were transferred after various times to E. coli OP50 plates. GFP expression was restored within 12 hours of transfer (FIG. 13E).

Because C50 carotenoids have multiple conjugated double bonds, they are likely to have antioxidant activity (Edge et al., 1997). However, it is unlikely that the ROS-quenching property of carotenoids is responsible for the suppression of pgp-5p::gfp induction for several reasons. First, pgp-5p::gfp is not induced by oxidative stress (Govindan et al., 2015). Second, whether known antioxidants can suppress the induction of pgp-5p::gfp was tested in a C. elegans translation defective mutant. eft-3(q145);pgp-5p::gfp animals grown on E. coli OP50 were treated with either N-acetyl cysteine, ascorbic acid, trolox or resveratrol and screened after 50 hours at 20° C. The induction of pgp-5p::gfp was not significantly different in animals treated with antioxidants compared to mock-treated eft-3(q145);pgp-5p::gfp animals (FIG. 14A). Third, commercially available carotenoids were tested for the ability to suppress the induction of pgp-5p::gfp in a C. elegans translation defective mutant. eft-3(q145);pgp-5p::gfp animals grown on E. coli OP50 were treated with either beta-carotene or astaxanthin and screened after 50 hours at 20° C. The induction of pgp-5p::gfp was not significantly different in animals fed on these antioxidants compared to mock-treated eft-3(q145);pgp-5p::gfp animals (FIG. 14B). Finally, eft-3(q145);pgp-5p::gfp animals were fed on E. coli expressing either zeaxanthin, neurosporene, violaxanthin, delta-carotene, or alpha-carotene and screened for GFP induction. The induction of pgp-5p::gfp was not significantly different in animals fed on E. coli expressing carotenoids compared to mock-treated eft-3(q145);pgp-5p::gfp animals (FIG. 14C). None of these carotenoids are C50 class carotenoids.

K. rhizophila also suppresses the detoxification response to translation inhibiting drugs. Hygromycin is a bacterially-produced antibiotic (from Streptomyces hygroscopicus) that inhibits translation and induces xenobiotic detoxification in C. elegans. While 10 μg/ml of hygromycin induces pgp-5p::gfp expression in animals fed E. coli OP50, animals fed K. rhizophila and 10 μg/ml of hygromycin failed to induce pgp-5p::gfp (FIG. 3A; FIG. 14D). However, at high concentrations of hygromycin, pgp-5p::gfp is induced both in animals fed on E. coli OP50 and K. rhizophila (FIG. 14D). By contrast, pgp-5p::gfp animals fed on K. rhizophila crtI(e10) or crtEb(e17) did not affect the GFP induction in response to hygromycin treatment (FIG. 3A). Similar results were obtained with emetine which blocks protein synthesis by binding to the 40S subunit of the ribosome. 6.25 g/ml of emetine induces pgp-5p::gfp expression in animals fed E. coli OP50 (FIGS. 15A&B); however, animals fed K. rhizophila and 6.25 g/ml of emetine fail to induce pgp-5p::gfp. However, at high concentrations of emetine, pgp-5p::gfp is induced both in animals fed E. coli OP50 and K. rhizophila (FIG. 15A). By contrast, pgp-5p::gfp animals fed K. rhizophila crtI(e10) or crtEb(e17) did not affect the GFP induction in response to emetine treatment (FIG. 15B). pgp-5p::gfp was activated in response to genotoxic stress induced by cisplatin, which interferes with DNA replication. While 1 mM cisplatin induces pgp-5p::gfp expression in animals fed E. coli OP50, animals fed K. rhizophila and 1 mM cisplatin fail to induce pgp-5p::gfp (FIG. 15C). By contrast, pgp-5p::gfp animals fed K. rhizophila crtI(e10), crtEb(e17), or crtYe(e22) did not affect the GFP induction in response to cisplatin treatment (FIG. 15C).

Because K. rhizophila carotenoids suppress the induction of C. elegans xenobiotic detoxification genes by translation defects, the ability of K. rhizophila carotenoids to increase C. elegans sensitivity to translational inhibitors was evaluated. While >80% of wildtype animals fed E. coli OP50 and 10 μg/ml hygromycin reach adulthood in four days, <10% of animals fed on E. coli OP50 and 10 μg/ml hygromycin and K. rhizophila carotenoid extract reach adulthood in four days (FIG. 3B; FIG. 15D). Carotenoids by themselves were not toxic to the worms in the absence of hygromycin (FIG. 3B; FIG. 15D). Similar results were obtained with emetine: Animals treated with K. rhizophila extracts were hypersensitive to emetine compared to animals fed on control extract (FIG. 3C). In addition, animals treated with K. rhizophila extracts were hypersensitive to cisplatin compared to animals fed on control extract (FIG. 3D). The xenobiotic hypersensitivity phenotype was not likely a generalized phenomenon because K. rhizophila carotenoid extract did not alter the sensitivity of animals to antimycin, a mitochondrial poison (FIG. 15E).

C. elegans food aversion behaviors are induced when animals are exposed to xenobiotics or essential gene inactivations (Melo and Ruvkun, 2012). Exposing animals to hygromycin (ribosomal translation inhibitor) or cisplatin (DNA replication inhibitor) induces strong food aversion. While ˜40% of animals exposed to 25 g/ml hygromycin display food aversion behavior, ˜15% of animals exposed to 25 g/ml hygromycin and K. rhizophila carotenoid extract show aversion (FIG. 3E). Similar results were obtained with cisplatin: ˜50% of animals exposed to 1 mM cisplatin display aversion behavior while <20% of animals exposed to 1 mM cisplatin and K. rhizophila carotenoid extract display food aversion (FIG. 3F).

Example 2. C. elegans Pathway Analysis of K. rhizophila Inhibition of Translational Surveillance

To assess how K. rhizophila carotenoids inhibit induction of xenobiotic detoxification response pathways, genetic epistasis analysis was conducted with a series of C. elegans mutations that disrupt or activate the signal transduction pathway for translational surveillance at various steps (Govindan et al., 2015). The zip-2/bZIP transcription factor is required for the induction of pgp-5p::gfp expression in response to translation inhibition and represents the last step in transcriptional induction (Govindan et al., 2015). Overexpression of ZIP-2::mCherry under a intestine-specific promoter was sufficient to induce pgp-5p::gfp expression in wildtype C. elegans even in the absence of translation inhibition (FIG. 4A). Whether K. rhizophila feeding affects pgp-5p::gfp induction was tested in a ZIP-2::mCherry overexpressing strain. pgp-5p::gfp induction was similar in animals fed on E. coli OP50 and K. rhizophila (FIG. 4A) suggesting that the K. rhizophila carotenoids disrupt a surveillance pathway component upstream of the ZIP-2 bZIP transcription factor (or a parallel pathway(s)).

The induction of C. elegans xenobiotic detoxification genes by translation inhibition is dependent on a bile acid signaling pathway (Govindan et al., 2015). C. elegans with genetic defects in bile acid biosynthesis fail to activate pgp-5p::gfp in response to eft-3(q145), RNAi of translational components, or G418 drug inhibition of translation, but that mammalian bile acids can reanimate this signal (Govindan et al., 2015). While K. rhizophila feeding inhibits the induction of pgp-5p::gfp in eft-3(q145) animals, addition of exogenous mammalian bile acids reactivates GFP expression even in the presence of wild type K. rhizophila (FIGS. 4B&C). Thus K. rhizophila carotenoids act either upstream or at the bile acid signaling step of this C. elegans translational surveillance and response pathway.

To determine the mechanistic pathways through which K. rhizophila carotenoids might modulate the bile acid signaling pathway, we conducted a cherry-picked RNAi screen of C. elegans homologues of eukaryotic genes that mediate carotenoid binding or transport (Table 1). In this screen, we fed eft-3(q145);pgp-5p::gfp animals on E. coli expressing dsRNA for the C. elegans homologue of carotenoid binding or transport proteins. When these animals reached adulthood, they were transferred to K. rhizophila seeded plates and scored for GFP induction after 24 hours. In animals fed on dsRNA negative control and transferred to K. rhizophila plates, pgp-5p::gfp expression was abrogated. Similar reduction of pgp-5p::gfp expression was found in the animals fed on 23 other dsRNA fed animals (Table 1). However, we found that in eft-3(q145);pgp-5p::gfp animals fed on lbp-5 dsRNA, pgp-5p::gfp expression was not suppressed (FIG. 4D). lbp-5 encodes an intracellular fatty acid binding protein that is predicted to function as a transporter of hydrophobic molecules such as lipids and steroid hormones (Xu et al., 2014).

TABLE 1 Known carotenoid binding proteins genes tested for suppression of gfp expression in eft-3(q145); pgp-5p::gfp animals in response to K. rhizophila RNAi on eft-3(q145); pgp-5::gfp, Transferred to Worm E. coli Gene Description OP50 Kocuria dsRNA control + − scav-6 ortholog of human SCARB1 (scavenger receptor class B member 1) F52F12.7 orthologous to the human gene STEROIDOGENIC + − ACUTE REGULATORY PROTEIN F25H2.6 ortholog of human COL4A3BP (collagen type IV alpha + − 3 binding protein) far-7 Fatty Acid/Retinol binding protein + − bcmo-2 ortholog of human RPE65 (RPE65, retinoid + − isomerohydrolase), BCO1 (beta-carotene oxygenase 1) and BCO2 (beta-carotene oxygenase 2) bcmo-1 ortholog of human RPE65 (RPE65, retinoid + − isomerohydrolase), BCO2 (beta-carotene oxygenase 2) and BCO1 (beta-carotene oxygenase 1) far-8 Fatty Acid/Retinol binding protein + − F45H7.2 a RhoGAP domain and a START (StAR-related lipid- + − transfer) domain F26F4.4 ortholog of human STARD3NL (STARD3 N-terminal + − like) and STARD3 (StAR related lipid transfer domain containing 3) lbp-5 predicted intracellular fatty acid binding protein (iFABP) + + that is most similar to the vertebrate muscle and heart FABPs lbp-6 ortholog of members of the human FABP (Fatty acid + − binding protein) family including FABP12 far-2 secreted fatty acid and retinol-binding proteins + − far-1 a fatty acid/retinol binding protein + − T28D6.7 ortholog of human STARD10 (StAR related lipid + − transfer domain containing 10) scav-3 SCAVenger receptor (C + D36 family) + − scav-2 ortholog of human CD36 antigen + − far-6 Fatty Acid/Retinol binding protein + − npa-1 strong binding protein for fatty acids and retinol + − (Vitamin A) C06H2.2 ortholog of human STARD7 (StAR related lipid transfer + − domain containing 7) atn-1 alpha-actinin homolog + − far-4 fatty acid and retinol-binding protein + − far-3 predicted to have lipid binding activity + − lbp-7 Predicted intracellular fatty acid binding protein (iFABP) + − scav-1 ortholog of human SCARB1 (scavenger receptor class B + − member 1) scav-5 ortholog of human SCARB1 (scavenger receptor class B + − member 1) scav-4 ortholog of human SCARB1 (scavenger receptor class B + − member 1) K02D3.2 human STARD4 (StAR related lipid transfer domain + − containing 4) +, GFP ON; −, GFP OFF

Further, to identify the eukaryotic cellular target(s) of the K. rhizophila carotenoid, a pull-down assay was conducted using rat liver cell extracts. Liver was chosen for identifying the carotenoid binding protein(s) for several reasons: First, it is the major site of xenobiotic detoxification. Second, it is the site of bile acid biosynthesis. Third, liver has a known carotenoid transport system. Fourth, large quantities of tissue can be easily obtained which was not feasible in C. elegans. To identify carotenoid binding proteins, protein extracts from rat liver were incubated with K. rhizophila carotenoid and unbound carotenoids were removed using size-exclusion chromatography. The carotenoid bound protein extract was subjected to anion-exchange chromatography and the protein fractions were eluted. The yellow fractions (indicating the presence of carotenoids) were pooled, concentrated and desalted. The concentrated peak was yellow-orange in color, indicating the presence of carotenoids was analyzed on native PAGE and the yellow-orange band visible was excised and mass spectrometry was conducted to identify proteins. 48 proteins were identified by mass spectrometry (Table 2). Interestingly, one of the proteins identified was FABP1 (Fatty acid binding protein 1), which is the homolog of C. elegans LBP-5. Among the proteins identified by mass spectrometry was PAK2 (p21 protein kinase), homolog of C. elegans pak-1, which we previously identified as a hit in the genome-wide RNAi screen required for pgp-5p::gfp induction in response to translation inhibition (Govindan et al., 2015). To determine whether any of these 48 proteins are required for pgp-5p::gfp induction in eft-3(q145);pgp-5p::gfp, an RNAi screen of the C. elegans homologs of these rat proteins was conducted. eft-3(q145);pgp-5p::gfp animals were fed on E. coli expressing dsRNA for the C. elegans homolog of each of the carotenoid binding proteins. When these animals reached adulthood, they were screened to determine whether they disrupted GFP induction. In this screen, RNAi of most of carotenoid binding proteins did not block pgp-5p::gfp induction in eft-3(q145);pgp-5p::gfp animals except for pak-1 RNAi (Table 2). Interestingly, one of the carotenoid binding proteins that was identified was AMACR (alpha-methylacyl-CoA Racemase), which is required for bile acid biosynthesis (Autio et al., 2014). In C. elegans, C24A3.4 and ZK892.4 are homologs of AMACR (FIG. 16A). While RNAi of C24A3.4 or ZK892.4 individually do not suppress the induction of pgp-5p::gfp in eft-3(q145);pgp-5p::gfp animals, double RNAi of C24A3.4 and ZK892.4 suppressed pgp-5p::gfp induction (FIG. 16B).

To determine whether any of these 48 C. elegans proteins are required for K. rhizophila carotenoid-induced suppression of pgp-5p::gfp induction, an RNAi screen was conducted of the C. elegans homologs of these proteins. When these animals reached adulthood, they were transferred to K. rhizophila seeded plates and scored for GFP induction after 24 hours. In animals fed on dsRNA negative control and transferred to K. rhizophila plates, pgp-5p::gfp expression was abrogated (Table 2). Similar reduction of pgp-5p::gfp expression was found in the animals fed in most dsRNA animals (Table 2); however, in animals treated with either chc-1 or fcho-1 or lbp-5 RNAi, pgp-5p::gfp induction was not abrogated (FIG. 4D). chc-1 encodes the C. elegans clathrin heavy chain ortholog while fcho-1 encodes the C. elegans homolog of F-BAR domain-containing fer/Cip4 homology domain-only (FCHo) family protein. Both CHC-1 and FCHO-1 mediate endocytic trafficking (Grant et al., 2006). An alternative model is that chc-1 or fcho-1 or lbp-5 RNAi by itself might induce pgp-5p::gfp expression even in the absence of ribosomal stress; however RNAi of these genes did not induce pgp-5p::gfp expression (FIG. 16C).

Without wishing to be bound the theory, based on the findings, a model of how K. rhizophila carotenoids inhibit xenobiotic detoxification response is proposed (FIG. 4E). Carotenoids released from K. rhizophila enter the C. elegans intestine via clathrin-mediated endocytosis. Within the intestinal cytoplasm, the carotenoids released from the endocytic vesicles are then bound by LBP-5 and delivered to the peroxisomes where it inhibits the bile acid biosynthesis via binding to AMACR. In addition, carotenoids also inhibit PAK-1 to disrupt xenobiotic induced upregulation of detoxification response.

TABLE 2 List of proteins identified in Mass spectrometry and effect of RNAi of their C. elegans homologs on the suppression of gfp expression in eft-3(q145); pgp-5p::gfp animals in response to K. rhizophila RNAi on eft- 3(q145); pgp-5::gfp, Transferred to Human Worm E. coli Total Protein Gene Description Sequencing OP50 Kocuria dsRNA control + − 15 PDIA5 D2092.4 Protein disulfide Verified + − isomerase family A 10 EHD3 rme-1 Plays a role in Verified + − endocytic transport 11 NUDC nud-1 Nuclear distribution C homolog 8 CLTC chc-1 Clathrin, heavy chain Verified + + (Hc) 6 METAP2 map-2 Methionyl Verified + − aminopeptidase 2 6 PAK2 pak-1 P21-activated kinase 2 Verified − − 6 EIF2S2 K04G2.1 Eukaryotic translation Verified + − initiation factor 6 RPS4X rps-4 Ribosomal protein S4, X-linked 5 2-Sep unc-59 Filament-forming Verified + − cytoskeletal GTPase 5 AMACR C24A3.4 alpha-methylacyl- Verified + − CoA racemase 5 ABCF1 F18E2.2 ATP-binding cassette, sub-family F 5 ACAD9 acdh-12 acyl-CoA Verified + − dehydrogenase 4 FCHO2 fcho-1 FCH domain only 2 Verified + + 4 PARVA pat-6 Parvin, alpha Verified + − 4 ELAC2 hoe-1 Zinc Verified + − phosphodiesterase 4 SIRT3 sir-2.1 Sirtuin 3 Verified + − 4 OSGEP Y71H2AM.1 O-sialoglycoprotein Verified + − endopeptidase 4 TAT tatn-1 Tyrosine Verified + − aminotransferase 4 PRKAB1 F55F3.1 Protein kinase, AMP- Verified + − activated 4 PDK2 ZK370.5 Pyruvate dehydrogenase kinase 4 MESDC2 bmy-1 Chaperone + − 7 7-Sep unc-59 Filament-forming Sequence + − cytoskeletal GTPase verified 4 ITSN1 itsn-1 Adapter protein; Sequence + − endocytosis verified 4 ARMC1 K05C4.7 Armadillo repeat Sequence + − containing 1 verified 4 ITIH4 No Inter-alpha-trypsin homolog inhibitor heavy chain 3 DNAJB11 dnj-20 DnaJ (Hsp40) No RNAi homolog, subfamily B clone 3 9-Sep unc-61 Filament-forming Sequence + − cytoskeletal GTPase verified 3 11-Sep unc-61 Filament-forming Sequence + − cytoskeletal GTPase verified 3 ITPA ZC395.7 Inosine triphosphatase Sequence + − verified 3 SLC9A3R1 nrfl-1 Solute carrier family 9 Sequence + − verified 3 FABP1 lbp-5 Fatty acid binding Sequence + + protein 1 verified 3 PRKAG1 aakg-1 Protein kinase, AMP- Sequence + − activated verified 3 DECR1 F53C11.3 2,4-dienoyl CoA No RNAi reductase 1, clone mitochondrial 3 DDX19A ddx-19 DEAD box Sequence + − polypeptide 19A verified 3 GDI1 gdi-1 GDP dissociation Sequence + − inhibitor 1 verified 3 MARS mars-1 methionyl-tRNA NoRNAi synthetase clone 3 FAM45A No Family with sequence homolog similarity 45 3 NAA15 Y50D7A.4 N(alpha)- Sequence + − acetyltransferase 15 verified 3 GRB2 sem-5 Growth factor Sequence + − receptor-bound verified protein 2 3 CAPN2 clp-1 Calcium-regulated Sequence + − non-lysosomal verified protease 3 CYFIP1 gex-2 Cytoplasmic FMR1 Sequence + − interacting protein 1 verified 3 HADH B0272.3 hydroxyacyl-CoA Sequence + − dehydrogenase verified 3 DDX46 F53H1.1 DEAD box; role in Sequence + − splicing verified 3 PUF60 rnp-6 poly-U binding Sequence + − splicing factor verified 3 AP3D1 apd-3 Adaptor; endocytosis Sequence + − verified 2 API5 No Antiapoptotic factor; Sequence + − homolog role in protein verified assembly 2 CRK Y41D4B.13 V-crk sarcoma virus Sequence + − CT10 oncogene verified homolog +, GFP ON; −, GFP OFF; no entry = not tested

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OTHER EMBODIMENTS

It is to be understood that while embodiments of the invention have been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. 

1. A method for the treatment of nausea and/or vomiting in a subject, the method comprising: administering to a subject in need thereof a therapeutically effective amount of a C50 carotenoid compound.
 2. The method of claim 1, wherein the subject has or is at risk of developing nausea and/or vomiting associated with chemotherapy or radiation.
 3. The method of claim 2, wherein the subject has or is at risk of developing chemotherapy-induced nausea and vomiting (CINV) or radiation-induced nausea and vomiting (RINV).
 4. The method of claim 1, wherein the subject has or is at risk of developing post-operative nausea and vomiting (PONV).
 5. The method of claim 1, wherein the C50 carotenoid compound is selected from the group consisting of decaprenoxanthin, C50-astaxanthin, C50-β-carotene, C50-carotene (n=3) (16,16-diisopentenylphytoene), C50-zeaxanthin, C50-caloxanthin, C50-nostoxanthin sarcinaxanthin, sarprenoxanthin, acyclic C50 carotenoid bacterioruberin, C50-canthaxanthin, C50-lycopene, C50-phytoene, and combinations thereof.
 6. The method of claim 5, wherein the C50 carotenoid compound is decaprenoxanthin.
 7. The method of claim 1, wherein the step of administering comprises administering a composition that is or comprises: (i) a C50-carotenoid-compound-synthesizing microbe or component thereof, (ii) a C50-carotenoid-compound-synthesizing microbe extract, (iii) an extracted carotenoid compound, or (iv) a combination thereof.
 8. The method of claim 7, wherein the C50-carotenoid-compound-synthesizing microbe is viable or alive.
 9. The method of claim 8, wherein the step of administering comprises administering a sufficient amount of the microbe to colonize the subject's microbiome.
 10. The method of claim 7, wherein the composition comprises or is prepared from a culture of the microbe.
 11. The method of claim 7, wherein the microbe is a strain that is found in nature.
 12. The method of claim 7, wherein the microbe is an engineered microbe.
 13. The method of claim 12, wherein the engineered microbe comprises a genetic alteration relative to an otherwise comparable reference microbe so that it produces the C50 carotenoid compound at an absolute or relative level different from that of the reference microbe.
 14. The method of claim 1, wherein the step of administering comprises administering a composition that comprises or delivers a synthesized C50 carotenoid compound.
 15. The method of claim 7, wherein the C50-carotenoid-compound-synthesizing microbe is selected from the group consisting of Kocuria rhizophila, Corynebacterium glutamicum, Arthrobacter arilaitensis, and combinations thereof.
 16. A method for the reduction of food aversion in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a C50 carotenoid compound.
 17. The method of claim 16, wherein the subject has or is at risk of developing nausea and vomiting associated with chemotherapy or radiation.
 18. The method of claim 17, wherein the subject has or is at risk of developing chemotherapy-induced nausea and vomiting (CINV) or radiation-induced nausea and vomiting (RINV).
 19. The method of claim 16, wherein the subject has or is at risk of developing post-operative nausea and vomiting (PONV).
 20. The method of claim 16, wherein the C50 carotenoid compound is selected from the group consisting of decaprenoxanthin, C50-astaxanthin, C50-β-carotene, C50-carotene (n=3) (16,16-diisopentenylphytoene), C50-zeaxanthin, C50-caloxanthin, C50-nostoxanthin sarcinaxanthin, sarprenoxanthin, acyclic C50 carotenoid bacterioruberin, C50-canthaxanthin, C50-lycopene, C50-phytoene, and combinations thereof.
 21. The method of claim 20, wherein the C50 carotenoid compound is decaprenoxanthin.
 22. The method of claim 16, wherein the step of administering comprises administering a composition that is or comprises: (i) a C50-carotenoid-compound-synthesizing microbe or component thereof, (ii) a C50-carotenoid-compound-synthesizing microbe extract, (iii) an extracted carotenoid compound, or (iv) a combination thereof.
 23. The method of claim 22, wherein the C50-carotenoid-compound-synthesizing microbe is viable or alive.
 24. The method of claim 23, wherein the step of administering comprises administering a sufficient amount of the microbe to colonize the subject's microbiome.
 25. The method of claim 22, wherein the composition comprises or is prepared from a culture of the microbe.
 26. The method of claim 22, wherein the microbe is a strain that is found in nature.
 27. The method of claim 22, wherein the microbe is an engineered microbe.
 28. The method of claim 27, wherein the engineered microbe comprises a genetic alteration relative to an otherwise comparable reference microbe so that it produces the C50 carotenoid compound at an absolute or relative level different from that of the reference microbe.
 29. The method of claim 16, wherein the step of administering comprises administering a composition that comprises or delivers a synthesized C50 carotenoid compound.
 30. The method of claim 22, wherein the C50-carotenoid-compound-synthesizing microbe is selected from the group consisting of Kocuria rhizophila, Corynebacterium glutamicum, Arthrobacter arilaitensis, and combinations thereof. 31.-74. (canceled) 